The present study investigated the role of actin polymerization and myosin motor protein activity in the gliding motility of Cryptosporidium parvum sporozoites. Short motility trails were detected using an indirect immunofluorescent assay (IFA) with a polyclonal antisporozoite antibody following incubation of sporozoites on poly-L-lysine-coated glass slides. Sporozoite motility was blocked following exposure to cytochalasin D, a myosin light-chain kinase inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexhydro-1,4-diazapin e, and the myosin ATPase inhibitor 2,3-butanedione monoxime. Sporozoites were observed to form rounded, blunt-ended shapes when exposed to these same inhibitors. Incubation of purified oocysts with these compounds did not significantly inhibit in vitro excystation or subsequent infectivity in cultured epithelial cells. Indirect IFA revealed a uniform distribution of actin protein throughout the body of the sporozoite; immunoelectron microscopy confirmed a diffuse intracellular pattern of gold particles in excysted sporozoites. Collectively, these findings show that sporozoite motility is dependent upon an intact actin-myosin motor system, and the dynamic interaction of F-actin and myosin motor proteins has a further role in maintaining the structural integrity of excysted sporozoites. Further, in vitro excystation and infectivity of C. parvum occurs in the absence of dynamic sporozoite locomotion.