The present study was designed to develop a procedure for purifying rPL-II and a homologous radioimmunoassay (RIA) for rPL-II. Molecular profiles of rPL-II were also investigated in tissue and plasma. rPL-II was purified 3,780-fold, based on its radioreceptor assay (RRA) activity compared to ovine prolactin (0PRL), from the placenta of day 18 pregnant rats using ammonium sulfate precipitation and chromatography on phenyl-Sepharose, DEAE-TOYOPEARL 650S, AF-chelate TOYOPEARL 650M and Sephadex G-100. Electrophoretic analysis on SDS gel revealed molecular weight heterogeneity of purified rPL-II, which consisted of three proteins; a major form with a molecular weight of 20.0 K and two minor forms with molecular weights of 20.6 K and 21.0 K under non-reducing conditions. One of the minor forms of rPL-II observed under non-reducing conditions disappeared with 2-mercaptoethanol treatment and the rest of the hormones migrated as 24.5 K and 25.0 K molecular weight species, suggesting that it is a cleaved form of rPL-II. Purified rPL-II displaced 125I-labelled oPRL from binding sites on rabbit mammary gland membranes in a dose-dependent manner. rPL-II and rPRL were, respectively, 21 and 2 per cent as effective as oPRL in the displacement. Antibody to purified rPL-II was raised in rabbits and a homologous RIA for rPL-II was developed. No displacement was observed with rPRL, rGH, oPRL, and other pituitary hormones up to 1,000 ng/ml. Molecular profiles of rPL-II in the placental tissue and plasma from day 18 pregnant rats were examined by gel chromatography on Sepharcryl S-300 HR and by Western blotting. Chromatography of the placental extracts revealed a single peak, which accounted for 86 per cent of the total RIA activity. Anti-rPL-II antiserum detected proteins of at least three molecular sizes as monomeric forms with molecular weights of 20.0, 20.6, and 21.0 K in the non-reducing placental extracts. One of them disappeared with 2-mercaptoethanol treatment and other two proteins had molecular weights of 24.5 and 25.0 K, indicating monomeric heterogeneity of rPL-II in the tissue. The elution profile of day 18 plasma in RIA activity gave two major peaks; the first, eluting just after the void volume (approximate molecular weight of 530 K) accounted for 35 per cent of the total RIA activity, and the second coinciding with the same elution volume as the monomeric form in the placental extract constituted about 26 per cent of the total RIA activity.(ABSTRACT TRUNCATED AT 400 WORDS)