The cytosolic P-enolpyruvate carboxykinase activity was measured in mammary gland and liver of animals at all stages of the reproductive cycle. The specific activity of this enzyme was almost absent in the mammary gland of virgin rats. Different pattern of P-enolpyruvate carboxykinase activity was obtained in liver and mammary gland during lactation cycle. The specific activity of the enzyme increased more than 40-fold in mammary gland and 2-fold in liver during the transition from mid-pregnancy to mid-lactation. Weaning of mid-lactating rats for 48 h resulted in an abrupt change in the enzyme activity in mammary gland while there was a small change in liver. In all the experiments performed, the activity of P-enolpyruvate correlates inversely with the plasma insulin levels described for the lactogenic process.