Multiple forms of PRL differing in their physicochemical and biological characteristics have been described. We have analyzed the molecular forms of human (h) PRL released in culture by pure hPRL-secreting tumors with a particular attention to glycosylated hPRL. The prolactinoma cells from six different tumors released, in serum-free conditions, 10-28 mg hPRL. The combination of polyacrylamide gel electrophoresis and immunoblotting techniques using a [125I]anti-hPRL monoclonal antibody allowed qualitative and quantitative analysis of the hPRL variants. The ratio of the glycosylated 25,000-mol wt form (G-hPRL) to the 23,000-mol wt nonglycosylated monomeric hPRL (NG-hPRL) varied from 0.13 to 0.25. Under the conditions of our studies, cleaved forms of the hormone (19,000 and 15,000 mol wt) accounted for less than 5% of the total immunoreactivity. G- and NG-hPRL were subsequently purified by gel filtration and lectin affinity chromatography. G-hPRL appeared fully sensitive to endoglycosidase F digestion, further supporting the presence of a freely accessible N-linked carbohydrate chain. When assayed for their ability to react with polyclonal antibodies directed against hPRL in a competitive RIA, G-hPRL was 3 times less immunoreactive than NG-hPRL. However, both types of hPRL exhibited superimposable displacement curves when tested in an immunoassay using an anti-hPRL monoclonal antibody. In binding studies using crude rabbit mammary gland membranes G-hPRL was half as potent as NG-hPRL. In stimulating the growth of the Nb2 lymphoma cell line, G-hPRL was 50% less active than NG-hPRL. Thus 1) under basal conditions, hPRL undergoes partial and variable glycosylation; 2) glycosylation of the hormone may modulate its immunoreactivity; 3) glycosylation of hPRL not only lowers its mammary gland receptor binding capacity but also its growth-promoting activity.