By using a specific heterologous double-antibody RIA, changes in the blood levels of PRL during pregnancy, pseudopregnancy, and lactation have been investigated for the first time in the rabbit. Blood levels fluctuate during early and midpregnancy in a manner similar to that in pseudopregnancy. Levels decline in the third trimester of pregnancy and increase dramatically (3- to 25-fold) at or 1--2 days before delivery. Pituitary levels of PRL showed no significant alteration, and fetal serum and amniotic fluid levels of PRL remain low (less than 10 ng/ml) throughout pregnancy. No significant PRL-like, GH-like, or placental lactogen-like activity could be demonstrated either in serum or in extracts of placenta (n = 262) taken between days 10 and 31 of pregnancy. Postpartum blood levels of PRL were similar in lactating and postpartum nonlactating females. In lactating females, suckling evoked an immediate increase (15- to 25-fold) in circulating PRL levels. Handling the female or the iv injection of oxytocin during lactation did not cause PRL release. In contrast, manual test stimulation caused an immediate increase in blood levels of PRL and a response pattern very similar to that of natural suckling. These results suggest that PRL release during suckling occurs solely in response to the tactile stimulation of the teats.