The effect of manual teat stimulation (milking paradigm) on release of prolactin was studied (1) in non-pregnant heifers (six each in age groups 3, 6, and 10 months), in 15 heifers at 100, 150, 200 and 250 days of gestation and at 30 and 90 days of lactation (during machine milking) and (3) simultaneously in six heifers at less than or equal to 100 and in six heifers at greater than or equal to 200 days of gestation. Among non-pregnant heifers small increases in prolactin response to teat stimulation were measured in three of six, two of six and none of six animals at 3, 6, or 10 months of age. In contrast, prolactin responses of the 15 heifers (area under response curves and absolute peaks) were significantly greater with each progressive stage of gestation. However, prolactin responses to machine milking at 30 days of lactation were significantly less than responses to manual teat stimulation during the latter stages of pregnancy, and responses at 90 days of lactation were further diminished. The changes in prolactin response during gestation were not due to seasonal effects as prolactin responses measured simultaneously in animals at different stages of gestation showed similar trends in responsiveness.