Serum prolactin concentrations were measured using a sensitive bioassay (Nb2 assay) and by radioimmunoassay in 11 patients with prolactin-secreting pituitary tumours (median serum immunoactive prolactin 5150 mU/L), and in 58 normal control subjects (median prolactin 190 mU/L). The mean ratio of serum prolactin bioactivity to immunoactivity was significantly lower in patients with prolactinomas than in normoprolactinaemic controls. Ten lactating women in the early post-partum period (median prolactin 3800 mU/L), studied as a model of physiological hyperprolactinaemia, also had reduced bioactivity to immunoactivity ratios. Overall, there was a significant negative correlation between bioactivity:immunoactivity ratio and serum immunoactive prolactin. The change in relative bioactivity of prolactin in serum samples from patients with prolactinomas and in women with physiological hyperprolactinaemia may reflect changes in the molecular heterogeneity of the hormone. Such changes may affect activity in both bioassays and radioimmunoassays.