|Cytokinins (CK) mediate a large spectrum of developmental and physiological regulations in plants. The aerial (shoot) and underground (root) portions of plant bodies reveal specific and sometimes even contrary responses to CK. However, the molecular mechanisms underlying the shoot and root specificity of CK-mediated regulations remain mostly unclear. Here we present results of a comprehensive approach in the study of the differential shoot and root CK response. We analyzed the time and tissue specificity of the CK response in 6-day-old seedlings treated with exogenous CK (5 uM 6-benzylaminopurine, BAP) in two time intervals: 30 min (designated as an “early” response) and 120 min of incubation (designated as a “delayed” CK response). Root and shoot tissues were collected and analyzed separately for both intervals and the specificity of CK response in both tissue types was analyzed on the level of proteome response, hormone metabolism and CK signalling. CK response at the proteome level was analyzed using 2-D gel electrophoresis with fluorescent SYPRO Ruby gel staining separately in both the shoot and the root and at both time intervals. Both qualitative (presence or absence of the respective spot) and quantitative changes in the protein abundance in comparison to the mock-treated controls were determined by image analysis of 2-D gels and individual proteins were identified by LC-MS/MS. Overall, we identified 43/18 differentially regulated proteins in the shoot and 31/21 differentially regulated proteins in the root in the early/delayed response, respectively. Comparison of our results with transcriptional regulations of subset of identified proteins show that tissue- and time-specific proteome changes are important mechanisms of an early CK response in Arabidopsis. We demonstrate that CK predominantly regulate proteins involved in carbohydrate and energy metabolism in the shoot and in protein synthesis and destination in the root. Further, we show that CK regulate plant hormonal metabolism, again in a tissue-specific way. In the root we have identified three out of four enzymes of the ethylene biosynthetic pathway to be upregulated in an early CK response, suggesting that majority of the ethylene biosynthetic pathway is under strong and immediate control of CK in the root. On the other hand, in the shoot we have identified several abscisic acid (ABA)-related proteins to be upregulated by CK. To further substantiate the potential crosstalk of BAP with metabolism of other hormones, we have analyzed changes of the endogenous hormone levels after CK application. In good accordance with our proteomic data, we found prompt upregulation of 1-aminocyclopropane-1-carboxylic acid, the rate-limiting precursor of ethylene biosynthesis in the root, while ABA was upregulated in the early response of the shoot. In addition, we found tissue-specific distribution of endogenous CK and their metabolites that revealed shoot- and root-specific responses to exogenous CK. Finally, we examined the contribution of CK-mediated ethylene production to MSP signaling during the early response and we present data suggesting that ethylene contributes to the output of multistep phosphorelay in the root, thus integrating both CK and ethylene signaling. In conclusion, we propose tissue-specific proteome response and CK-mediated hormonal crosstalk as important factors contributing to the long-known specificity of CK regulations over shoot and root development.