During apoptosis several mitochondrial proteins are released. They participate in caspase-independent nuclear DNA degradation, especially apoptosis-inducing factor (AIF, also PDCD8 or AIFM1) and endonuclease G. Another interesting protein, which was expected to act similarly as AIF due to the high sequence homology with AIF is AIF-homologous mitochondrion-associated inducer of death (AMID, also PRG3 or AIFM2). We studied the cellular localization and colocalization of proteins AIF, endonuclease G and AMID experimentally using vectors encoding proteins of interest fused to the fluorescent proteins and using bioinformatic predictions, that analyze the amino acid sequence of the proteins with various algorithms. We confirmed the colocalization of AIF and endonuclease G in the mitochondria of human cells. AMID was found to be the cytoplasmic protein, bound to various cellular surfaces from their cytoplasmic side. Overexpression of fusion protein AMID-HcRed-tandem was not lethal to the cells or mitochondria. We did not observe its translocation into the nucleus during apoptosis. The proposed role of AMID in apoptosis was thus not observed. Bioinformatic predictions and time-lapse FRET experiments were conducted to analyze the interactions of the studied proteins with each other and with other possible ligands in living and fixed human cells. We conducted molecular modelling of proteins with not yet recognized 3D structure and these models were then employed in molecular docking simulations of interactions of studied proteins. Our results contribute to the comprehension of localization, interactions and functions of AMID, AIF, and endonuclease G in human cells. This work was supported by the Grant Agency of the Czech Republic (grant number 204/05/P090) and by The Ministry of Education, Youth and Sports of the Czech Republic (projects number MSM0021622419 and LC535).