Within the genus Cryptosporidium 2 lineages have evolved, one adapted to the acid environment of the stomach and abomasum, the other comprising parasites that multiply in the small intestine. We tested whether the release of sporozoites from oocysts, a process known as excystation, is triggered by conditions which mimic the site of infection. Specifically, we exposed oocysts from gastric and intestinal Cryptosporidium species to acid conditions or to a neutral solution of taurocholic acid, at 37°C. We found that oocysts from the gastric species C. muris and C. andersoni excysted in both conditions, whereas the intestinal species C. parvum and C. hominis did not respond to acid. When the effect of temperature alone was tested on C. muris and C. parvum, only oocysts from the former species excysted in significant numbers. Oocysts from intestinal species did not respond to temperature alone, nor to acidity. These observations are consistent with the need of gastric species to rapidly excyst and release the sporozoites upon ingestion, and indicate that Cryptosporidium oocysts have evolved to maximize delivery of sporozoites to the region of the gastro-intestinal tract where the parasite multiplies.