DNA synthesis and DNA polymerase-alpha, -beta and -gamma activities in the rabbit mammary gland were studied during hormone-directed cellular growth. It was found that during pregnancy, early lactation and after injection of prolactin, changes in the activity of DNA polymerase-alpha paralleled the rate of mammary gland DNA synthesis. It was also found that the amount of polymerase-alpha activity bound to isolated chromatin depended on the physiological state of the animal. During pregnancy and early lactation changes in the activity of chromatin-bound enzyme correlated directly with the rate of DNA synthesis (r = 0.83). Moreover, in virgin rabbits treated with prolactin the activity of chromatin-bound DNA polymerase-alpha increased markedly at the same time as the DNA-synthetic rate increased. No correlation of the DNA-synthetic rate was found with the activity of soluble (cytosolic) DNA polymerase-alpha or with the activity of soluble or chromatin-bound DNA polymerases-beta and -gamma. On the basis of these results it is suggested that in the developing mammary gland both the activity and cellular distribution of DNA polymerase-alpha might be subject to hormonal regulation.