Prolactin present in maternal blood, fetal blood, and amniotic fluid samples obtained simultaneously at term was examined. The relative concentrations of prolactin in maternal and fetal serum samples were found to vary with each individual case. However, amniotic fluid samples contained a much higher level of prolactin than did both maternal and fetal serum samples in every case. After gel filtration, three separate peaks of immunoreactive prolactin were observed, probably representing three different sizes of the hormone molecule. The main component was small prolactin, constituting at least 69% of the total prolactin immunoreactivity in blood samples and 90% in amniotic fluids. Prolactin was found to be very similar in cord venous and arterial sera, both quantitatively and in terms of heterogeneity. There were more noticeable differences in the percentages of medium and small prolactin present in serum samples than in amniotic fluid samples. Big prolactin was present in all samples; its percentage in maternal serum was slightly higher than its percentages in the other samples. These results suggest that: 1) The mother and fetus carry on independent secretion of prolactin during gestation. 2) The size heterogeneity of prolactin in amniotic fluid differs from maternal hPRL to a greater degree than it does from fetal hPRL. 3) The prolactin in amniotic fluid appears to be of fetal origin.