Four isoforms of glycosylated prolactin (G-pPRL) were isolated from porcine pituitary glands by affinity chromatography and concanavalin A-Sepharose, based upon differences in their affinity for the lectin. Structural analysis indicated differences in the carbohydrate units of the four G-pPRLs. N-glycanase treatment cleaved the oligosaccharide from the G-pPRLs, establishing N-linked glycosylation. The binding of G-pPRLs to receptors from lactating rabbit mammary glands was only 3-8% that of nonglycosylated pPRL (NG-pPRL). The immunological crossreactivity of the G-pPRLs varied from 36 to 65% that of NG-pPRL. When tested in the pigeon crop sac bioassay, G-pPRLs were only 11-40% as active as NG-pPRL. The metabolic clearance rate of one of the G-pPRLs was slower and another faster than that of NG-pPRL. We conclude that there are several forms of G-PRL of variable immuno- and bio-potencies in the porcine pituitary, and that the current radioimmunoassay for the hormone does not measure the actual bioactivity.