Maize beta-glucosidase Zm-p60.1 is one of many enzymes which are important for plant development. It liberates free zeatin from its transport and/or storage form zeatin-O-glucoside. Using an adapted site specific non-saturated random mutagenesis approach, it were prepared five multi-site mutants surrounding the active site (W373K/M376L, W373K/P372S/M376L, W373K/P372T/M376L, W373K/P372S and W373K/P372T) derived from the single mutant W373K to study the effect(s) of amino-acid changes on substrate specificity towards natural (trans-zeatin-O-beta-D-glucopyranoside and cis-zeatin-O-beta-D-glucopyranoside) and artificial (4-nitorophenyl-O-beta-D-glucopyranoside and 4-methylumbellyferyl O-beta-D-glucopyranoside) substrates. Kinetic and substrate specificity studies confirmed large differences among set of mutated enzymes. All enzymes surprisingly preferred cis-zeatin-O-beta-D-glucopyranoside over trans-zeatin-O-beta-D-glucopyranoside, whereas differences in hydrolytic efficiencies are considerable. Quantitative TLC confirmed the best cZOG/tZOG hydrolysis ratio toward cis-zeatin-O-beta-D-glucopyranoside in the triple mutant W373K/P372T/M376L. Moreover, it was also confirmed that only wild-type hydrolyzed trans-zeatin-N9-beta-D-glucopyranoside. No known plant beta-glucosidase hydrolyzes this substrate. Hydrolysis of trans-zeatin-N7-beta-D-glucopyranoside was not observed at all.