Videomicroscopy and transmission electron microscopy were used to study the interaction of Toxoplasma gondii sporozoites with cultured cardiopulmonary artery endothelial, embryonic bovine tracheal and Madin-Darby bovine kidney cells. No moving junction or exocytosis of rhoptries, micronemes, and dense granules was detected during the initial penetration of sporozoites into cultured cells, whereas constriction of the sporozoite and partial exocytosis of rhoptries occurred during movement of the sporozoite from the first parasitophorous vacuole (PV1) into the second vacuole (PV2). The PV1 was unusually large, lacked a tubulovesicular membrane network (TMN), and had an indistinct parasitophorous vacuolar membrane (PVM). Comparatively, the PV2 was small, had a distinct PVM, contained a well-developed TMN, and was surrounded by numerous host cell mitochondria. Sporozoites that passed completely through cells carried with them an envelope of host cell membranes and cytoplasm. Cultured cells occasionally endocytosed sporozoites that were enveloped by host cell material. After formation of the PV2, sporozoites replicated by endodyogeny to form tachyzoites.