Cystoisospora canis is a coccidial parasite of the intestinal tract that can cause severe disease in dogs. Clinical signs include watery diarrhea, vomiting, fever, and weight loss. Extraintestinal stages of Cystoisospora spp. have been demonstrated in the mesenteric lymph nodes of paratenic hosts. Information on the biology of extraintestinal stages of canine Cystoisospora species is limited. The current study examined the development of C. canis in 2 noncanine cell lines and the ultrastructure of the monozoic cysts that formed. Monolayers of bovine turbinate cells and African green monkey kidney cells were grown on coverslips and inoculated with excysted C. canis sporozoites. Coverslips were collected on various days and fixed and stained for light microscopy (LM) or transmission electron microscopy (TEM). A single, centrally located, slightly crescent-shaped sporozoite surrounded by a thick cyst wall within a parasitophorous vacuole was observed with the use of LM and TEM. No division and no multinucleated stages were observed with either LM or TEM. With TEM, typical organelles of sporozoites were observed, such as rhoptries, dense granules, a crystalloid body, polysaccharide granules, and a conoid. The structure and ultrastructure of C. canis monozoic cysts produced in vitro are similar to extraintestinal cysts of other Cystoisospora species in experimentally infected animals and those of Cystoisospora belli observed in immunocompromised humans. This is the first study that fully demonstrates in vitro the development of what structurally resemble extraintestinal cysts of a Cystoisospora spp.