Cyclic AMP was measured in guinea pig mammary gland biopsies from midpregnancy through lactation and in daily milk samples throughout lactation. The results indicate that mammary gland cAMP levels rise sharply in late pregnancy to a prepartum peak and then drop abruptly at partus. This is similar to the pattern observed by others in rat and mouse although the guinea pig does not undergo a prepartum progesterone withdrawal. In animals with a 3-wk lactation period, milk cAMP concentration decreases approximately 40% between days 10 and 20, whereas the mammary gland level increases 22% per unit wet tissue weight. Although milk concentrations of lactose and cAMP are weakly correlated, a strong correlation exists between the total collected daily outputs of these two substances. The results suggest that, as lactation proceeds, mammary gland cAMP levels gradually increase, whereas milk cAMP levels decrease. Concurrently, lactose output decreases as lactation proceeds. These observations are consistent with first, a milk secretory route for regulating mammary gland cAMP levels (in addition to synthesis and hydrolysis), and second, an inhibitory action of cAMP on lactose synthesis.