The effects of hypophysectomy, adrenalectomy, alloxan diabetes, ovariectomy and appropriate hormone therapies upon enzyme activities in lactating rat mammary glands were investigated. Alloxan diabetes and ovariectomy did not appreciably affect enzyme patterns. Treatment of ovariectomized animals with estrogen increased levels of glucose-6-P dehydrogenase and fatty acid synthetase. Hypophysectomy resulted in depressions in the activities of glucose-6-P dehydrogenase, 6-P-gluconate dehydrogenase, fatty acid synthetase, UDPG pyrophosphorylase, P-glucose mutase, malic enzyme and citrate cleavage enzyme. The levels of a number of other enzymes were unaffected. Treatment with cortisol partially maintained activities of UDPG pyrophosphorylase, citrate cleavage enzyme and malic enzyme. Treatment with prolactin partially maintained malic enzyme activities. Treatment with cortisol plus prolactin maintained essentially normal enzyme levels. Adrenalectomy and cortisol treatment affected the activities of glucose-6-P dehydrogenase, fatty acid synthetase, UDPG pyrophosphorylase and citrate cleavage enzyme. On the basis of these and previous observations, it is concluded that cortisol regulates UDPG pyrophosphorylase and citrate cleavage enzyme synthesis and that both cortisol and prolactin are involved in the regulation of glucose-6-P dehydrogenase, 6-P-gluconate dehydrogenase, P-glucose mutase and fatty acid synthetase synthesis.