The present study describes the complete in vitro development of Cryptosporidium parvum (cattle genotype) in RPMI-1640 maintenance medium devoid of host cells. This represents the first report in which Cryptosporidium is shown to multiply, develop and complete its life cycle without the need for host cells. Furthermore, cultivation of Cryptosporidium in diphasic medium consisting of a coagulated new born calf serum base overlaid with maintenance medium greatly increased the total number of Cryptosporidium stages. Type I and II meronts were detected giving rise to two morphologically different merozoites. Type I meronts, which appear as grape-like clusters as early as 48 h post culture inoculation, release merozoites, which are actively motile, and circular to oval in shape. Type II meronts group in a rosette-like pattern and could not be detected until day 3 of culturing. Most of the merozoites released from type II meronts are generally spindle-shaped with pointed ends, while others are rounded or pleomorphic. In contrast to type I, merozoites from type II meronts are less active and larger in size. Sexual stages (micro and macrogamonts) were observed within 6-7 days of culturing. Microgamonts were darker than macrogamonts, with developing microgametes, which could be seen accumulating at the periphery. Macrogamonts have a characteristic peripheral nucleus and smooth outer surface. Oocysts at different levels of sporulation were seen 8 days post culture inoculation. Cultures were terminated after 4 months when the C. parvum life cycle was still being perpetuated with the presence of large numbers of excysting and intact oocysts. Culture-derived oocysts obtained after 46 days p.i. were infective to 7- to 8-day-old ARC/Swiss mice. The impact of C. parvum developing in cell-free culture is very significant and will facilitate many aspects of Cryptosporidium research.