The pathogenic yeast Candida albicans can undergo a dramatic change in morphology from round yeast cells to long filamentous cells called hyphae. We have cloned the CaMYO5 gene encoding the only myosin I in C. albicans. A strain with a deletion of both copies of CaMYO5 is viable but cannot form hyphae under all hypha-inducing conditions tested. This mutant exhibits a higher frequency of random budding and a depolarized distribution of cortical actin patches relative to the wild-type strain. We found that polar budding, polarized localization of cortical actin patches, and hypha formation are dependent on a specific phosphorylation site on myosin I, called the "TEDS-rule" site. Mutation of this serine 366 to alanine gives rise to the null mutant phenotype, while a S366D mutation, the product of which mimics a phosphorylated serine, allows hypha formation. However, the S366D mutation still causes a depolarized distribution of cortical actin patches in budding cells, similar to that in the null mutant. The localization of CaMyo5-GFP together with cortical actin patches at the bud and hyphal tips is also dependent on serine 366. Intriguingly, the cortical actin patches in the majority of the hyphae of the mutant expressing Camyo5(S366D) were depolarized, suggesting that although their distribution is dependent on myosin I localization, polarized cortical actin patches may not be required for hypha formation.