Two forms of highly purified prolactin (PRL) were obtained from pituitary glands of Xenopus laevis by extraction of acetone-dried powder with acid acetone and high-performance liquid chromatography on anion exchange, gel filtration, and reverse-phase columns. Purification was monitored by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis employing antiserum against bullfrog PRL. The Xenopus prolactins (xPRL-I and xPRL-II) thus obtained were shown to have similar molecular weights of 23,000 as determined by SDS-PAGE. The isoelectric points of xPRL-I and xPRL-II determined by isoelectric focusing were 5.6 and 5.3, respectively. Both hormones blocked T4-induced shrinkage of Xenopus tadpole tail fin in vitro. The amino acid compositions of the xPRLs resembled that of bullfrog PRL. The partial amino acid sequences of xPRL-I and of xPRL-II showed 78 and 68% homology with the comparable portion of the sequence of bullfrog PRL, respectively. Homology between xPRL-I and xPRL-II was 90%.