AbstractThe family of myosin motors is comprised of numerous classes distributed among a diverse set of organisms and cell types. We have identified an unconventional myosin gene (MYO3) in the yeastSaccharomyces cerevisiaeand show that it is member of a subclass of unconventional myosin proteins originally found only in the amoeboid organismsDictyosteliumandAcanthamoeba.Identification of this protein in these genetically and morphologically divergent organisms suggests that it will be ubiquitous in eukaryotes and that it has a role in the basic functions of the eukaryotic cell. We have constructed a strain of yeast missing 99% of the MYO3 coding sequence. This mutation has no observable phenotypic effect, placing MYO3 into a growing class of yeast genes which are dispensable under laboratory conditions, perhaps due to genetic redundancy. Alignment of MYO3 with other unconventional myosins shows that it shares with a subset of them a previously unrecognized region of homology in the tail; this region falls within a domain identified as important for mediating nonspecific electrostatic interactions with membranes. The existence of this region suggests that it may be involved in mediating specific protein‐protein interactions, possibly helping to localize this myosin to specific membranes or membrane regions. In addition, we show that “classic” myosin I proteins share a region of hyper‐proline‐richness 10 amino acids before the SH3 domain. Proline‐rich regions have recently been implicated as SH3 binding sites, which suggests that this region might be involved with regulating or in other ways interacting with SH3 domains. © 1995 Wil