We have investigated the minimal requirements of the tail region for myosin I function in vivo using the filamentous fungus Aspergillus nidulans. The CL3 strain (McGoldrick, C. A., Gruver, C., and May, G. S. (1995) J. Cell Biol. 128, 577-587) was transformed with a variety of myoA constructs containing mutations in the IQ, TH-1-like, SH3, and proline-rich domains by frameshift or in-frame deletions of the tail domains. The resulting strains contained wild type myoA driven by the alcA promoter and a mutant myoA driven by its endogenous promoter. This strategy allowed for selective expression of the wild type and/or mutant form of MYOA by the choice of growth medium. Proper septation and hyphal branching were found to be dependent on the interaction of the IQ motifs with calmodulin, as well as, the presence of its proline-rich domain. Additionally, a single proline-rich motif was sufficient for nearly wild type MYOA function. Most surprisingly, the SH3 domain was not essential for MYOA function. These studies expand our previous knowledge of the function of MYOA to include roles in hyphal morphogenesis, septal wall formation, and cell polarity, laying the groundwork for more detailed investigations on the function of the various tail domains in MYOA.