The motor properties of the two yeast class V myosins, Myo2p and Myo4p, were examined using in vitro motility assays. Both myosins are active motors with maximum velocities of 4.5 microm/s for Myo2p and 1.1 microm/s for Myo4p. Myo2p motility is Ca(2+) insensitive. Both myosins have properties of a nonprocessive motor, unlike chick myosin-Va (M5a), which behaves as a processive motor when assayed under identical conditions. Additional support for the idea that Myo2p is a nonprocessive motor comes from actin cosedimentation assays, which show that Myo2p has a low affinity for F-actin in the presence of ATP and Ca(2+), unlike chick brain M5a. These studies suggest that if Myo2p functions in organelle transport, at least five molecules of Myo2p must be present per organelle to promote directed movement.