Two molecular forms of prolactin (PRL), glycosylated and non-glycosylated, were isolated from pituitary glands of two reptiles, alligator and crocodile. The reptilian PRLs were extracted under alkaline conditions from the precipitate obtained after pituitaries were first extracted with 0.25 M sucrose, 1 mM NH4HCO3, pH 6.3. Purification was performed by ion exchange chromatography on DE-52, gel filtration on Sephadex G-75 superfine, and reversed phase high performance liquid chromatography. Two forms of both alligator and crocodile PRL, designated PRLI and PRLII, with molecular weights of 26,000 and 24,000 were isolated. Alligator and crocodile PRLI and PRLII were stained specifically in immunoblots with anti-sea turtle PRL and anti-ostrich PRL. Sequence analysis revealed that both forms of alligator and crocodile PRLs consisted of 199 amino acid residues with a glycosylation consensus sequence (Asn-Ala-Ser) at position 60 in alligator and crocodile PRLs with a molecular weight of 26,000 (PRLI). In contrast, Thr was substituted for Asn at position 60 in the PRLs with a molecular weight of 24,000 (PRLII). The sequences of alligator PRLs differed from crocodile PRLs only in position 134: Val for alligator PRLs and Ile for crocodile PRLs. There is a high degree of structural conservation between the reptilian PRLs isolated in this study and avian PRL; each showed 92% sequence identity with chicken PRL and 89% with turkey PRL.