Six canine mammary tumor cell lines (5 epithelial, 1 myoepithelial) were established from dogs with spontaneous mammary carcinomas. The cells were characterized for features of morphology and growth patterns on plastic, ultrastructure, cloning efficiency in soft agar, tumorigenicity in athymic nude mice, and hormonal receptor status. Biologic properties of the cell lines were distinct and preserved during long-term culture in vitro. All cell lines were clonogenic, 5 of 6 were tumorigenic, and 3 of 5 were receptor positive for estrogen and/or progesterone.