To assess the viability of the oocysts from Cryptosporidium sp, an in vitro excystation technique was developed and optimized. To this end, oocysts from Cryptosporidium sp were isolated from a sample of fecal material from a child with symptomatic cryptosporidiosis. The specimen was processed by a discontinuous-sucrose-gradient technique yielding a final suspension of purified and concentrated oocysts. The resulting oocysts were quantified by counting an aliquot in a Neubauer hemocytometer chamber and the remainder were preserved in phosphate-buffered saline at pH 7.2 and 4 degrees C. The two protocols tested through the use of 100-microliter aliquots containing 178,000 oocytes/mm3 were: I. Treatment at 37 degrees C for 24 h with 1% (v/v) bile plus 0.44% (w/v) sodium bicarbonate. Group A: pretreated with acidified trypsin (pH 2.75) at 37 degrees C for 1 h; Group B: not pretreated. II. Treatment at 37 degrees C for 24 h with 1% (v/v) bile at pH 6 and at pH 8 either with or without 0.44% (w/v) sodium bicarbonate in air or an atmosphere containing 10% (v/v) CO2. Group 1: bile, pH 6; Group 2: bile, pH 6 plus CO2; Group 3: bile, pH 6 plus bicarbonate; Group 4: bile, pH 8 plus bicarbonate; Group 5: bile, pH 6 plus bicarbonate plus CO2; Group 6: bile, pH 8 plus bicarbonate plus CO2; Group 7: bile, pH 8; and Group 8: bile, pH 8 plus CO2. We then assessed the degree of excystation in all the groups by counting the number of intact oocysts in the hemocytometer. For the first protocol, the oocyst count after excystation revealed an average of 28,000 oocysts/mm3 in Group A and 22,000 in Group B. These values indicate excystation frequencies of 84.3% and 87.6% for these two groups, respectively. The corresponding excystation percentages for groups 1-8 of protocol II were 49.5, 56.7, 83.2, 88.3, 88.3, 92.7, and 94.1, respectively. Preincubation with trypsin failed to augment oocyst excystation in Cryptosporidium in any of these experimental circumstances. The optimal conditions for oocyst excystation in these 24 h incubations were thus seen to be in the presence of 1% (v/v) bile at pH 8, and 37 degrees C under an atmosphere of 10% (v/v) CO2.