SUMMARYAnin vitrobioassay for lactogenic hormones, based on the specific mitogenic effects of lactogens on cultured Nb2 node rat lymphoma cells, was used to measure the levels of lactogens in whole serum and in fractionated serum samples from three patients with prolactin (PRL) secreting pituitary adenomas. Under basal conditions, after pituitary stimulation, and following treatment with bromocriptine which greatly decreased the serum PRL levels, the bioassay (BA) results closely paralleled fluctuations in the sum of radioimmunoassay (RIA) estimates of serum PRL and growth hormone concentrations. The extreme sensitivity (10 pg/ml) of the BA facilitated measurement of PRL in fractions obtained after Sephadex G‐100 chromatography of only 0·1 to 0·3 ml of sera from both untreated and bromocriptine‐treated prolactinoma patients. In both types of samples, similar heterogenous patterns of bioactive PRL were observed, although most of the recovered activity appeared to be due to monomeric PRL. BA and RIA of fractions obtained after DEAE cellulose chromatography of sera from untreated prolactinoma patients revealed two PRL peaks, the first one containing 75% of the activity. Therapy with bromocriptine led to a reduction in the total serum PRL activity and, in particular, in the activity of the second