Although pituitary prolactin (PRL) exhibits size and charge variability, questions concerning the structural heterogeneity and biological potency of hormonal forms secreted in vivo remain. In the present studies, monomeric PRL in male rat pituitaries and plasma was fractionated by Sephacryl S-100 size exclusion chromatography and aqueous chromatofocusing to resolve size and charge forms under conditions compatible with optimum preservation of biological activity. Individual hormonal variants were subsequently evaluated for their ability to stimulate the growth of PRL receptor-bearing rat lymphoma cells in vitro. Pituitary elution profiles contained several cross-reactive size variants ranging from 30.4 to 21.5 kD in M(r); major hormonal peaks were eluted at 25.6, 24.3, and 23.6 kD. Multiple size forms of PRL were also detected in plasma profiles, with predominant peaks eluting between 26.5 and 21.5 kD M(r) in size. Mean B/I ratios, established as an index of relative biopotency, varied significantly between size variants obtained from pituitary and plasma. Pituitary PRL size variants of 27.1, 24.3, and 21.5 kD exhibited greatest potency in the in vitro bioassay, whereas the 25.6- and 23.6-kD forms were least potent under these conditions. Of the PRL size variants detected in peripheral plasma, those of 24.3 and 21.5 kD size were characterized by highest mean B/I ratios. Pituitary 24.3-kD PRL was chromatofocused as five charge variants of pI 5.34, 5.31, 5.26, 5.20, and 5.14; only some of these isomers are apparently secreted in vivo, since pI values for plasma charge isomers ranged from 5.26 to 5.14. Charge isomers of pituitary 24.3-kD PRL exerted variable mitogenicity in the Nb2 in vitro bioassay. The highest mean B/I ratio was associated with the relatively basic 24.3-kD isomer of pI 5.31; the relative biopotency of more acidic charge isomers was progressively diminished with increasing acidic charge. In summary, the present findings show that both size and charge variants of male rat pituitary PRL exhibit differential biopotency in vitro, indicative of their functional heterogeneity. The demonstration of multiple hormonal forms in peripheral plasma suggests that the net biopotency of circulating PRL reflects the sum of activity of structurally and functionally diverse molecules.