Ypt/Rab GTPases are key regulators of vesicular transport. One or more Ypts identify the interface of each membrane organelle. The functional pair of trans-Golgi GTPases Ypt31/32 is required for the formation of secretory vesicles. Myosins are required for the motility of a number of organelles. In yeast, type V myosin Myo2 attaches to secretory vesicles through its globulat-tail domain (GTD) and facilitates their polarized delivery to the sites of cell growth, represented by the bud. Examples of Ypt/Rab GTPases recruiting the motor protein myosin to membranes of a specific organelle have been reported. Here, we identify Myo2 as an effector of Ypt31/32 and show that disruption of Myo2-Ypt31/32 interaction results in defective polarized secretion. We show that Myo2-Ypt31/32 interaction is specific, direct and restricted to GTPbound form of Ypt31/32, using the yeast two-hybrid system and co-precipitation of recombinant proteins. The physiological relevance of this interaction is shown by genetic interactions between MYO2 and YPT31/32 and co-localization of the proteins. We show the physiological and mechanistic function of this interaction by determining phenotypes of myo2 mutant alleles that disrupts this interaction. We demonstrate that this interaction is important for viability, for polarized delivery of secretory vesicles and for proper intra-cellular localization of both Myo2 and Ypt31/32. However, it is questionable whether Myo2-Ypt31/32 interaction is required for recruiting myosin to secretory vesicles.