In vitro excystation studies were done on the metacercarial cysts of Ribeiroia ondatrae obtained from naturally infected leopard frogs, Rana pipiens. Studies were done in Petri dish cultures in various media maintained at 41 degrees C. The media consisted of 0.5% trypsin-0.5% bile salts in Earle's balanced salt solution at pH 7.8 (TB), 1% acid pepsin (AP) in 0.85% saline adjusted to pH 2 with HCl; acid saline (AS) adjusted to pH 2 with HCl, and saline (S). The TB medium, used successfully to excyst the metacercariae of numerous species of digeneans, did not induce excystation of Ribeiroia ondatrae. Some excystation occurred in AS but not in S. Excystation was 100% in AP with most organisms excysting within 30 s after placement in the medium at 41 degrees C. Excystation in vitro in AP at 41 degrees C is compatible with the fact that this organism establishes itself in the acid-pepsin environment of the proventriculus of avian hosts.