In order to detect putative markers of prolactin-secreting pituitary tumours, adult rats were subjected to long-term oestrogenization with oestradiol benzoate (OE2) on a monthly basis. At 6 months, anterior pituitaries were dissected and incubated either as tissue fragments or as dispersed cells with a [35S]methionine mix for labelling. Proteins released into the incubation medium and from tissue extracts were further analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and fluorography. Oestrogen induced the appearance in the incubation medium of a protein (OE2 band) with an M(r) of 38,000 under reducing conditions, and high specific activity. Surprisingly, such a protein was not detected in tissue extracts. The OE2 band was detectable by 7 days after the first dose of oestrogen, and remained throughout 1 year of treatment. The tumour cell line GH3 showed a similar OE2 band which was further enhanced by oestrogens. The protein was observed similarly in both female and male pituitary donors, either intact or gonadectomized, and also in rats of different strains, suggesting that its appearance was independent of the strain of rat and gonadal status. Furthermore, the OE2 band was specific for pituitary cells and not produced by other oestrogenized tissues. No alteration in the rate of generation or the electrophoretic pattern of the OE2 band was observed when pituitary cells from oestrogenized rats were metabolically labelled while being incubated with tunicamycin. Furthermore, a system for glycan detection, adsorption to Concanavalin A or incubation with endoglycosidase F also failed to show a clear amount of glycosylation of the oestrogen-induced protein. Both immunoprecipitation experiments and time-limited proteolysis with V8 protease ruled out the possibility that the OE2 band could be structurally related to either GH or prolactin. In conclusion, oestrogens induce the generation of a new monocatenary protein with an apparent M(r) of 38,000, which has at least one intramolecular disulphide loop and is not glycosylated. The OE2 band was detected only in incubation medium and never in tissue extracts.