Poly(adenosine diphosphate [ADPI-ribosyl)ation, although associated with differentiation in many systems. exhibited a reciprocal relationship with mammary gland differentiation, and both the synthetic and degradatory pathways complemented each other in this regard. Poly(ADP-ribosy1)synthetase activity declined during pregnancy and lactation, while poly(ADP4bose) degradatory activity rose late in pregnancy and peaked during lactation. In explant cultures, similar changes occurred and appeared to be under separate hormonal control; prolactin suppressed the synthetase activity, whereas insulin stimulated the poly(ADP-ribo-sy1)glycohydrolase activity. This latter effect may be mediated by a decline in cAMP levels for the following reasons: the glycohydrolase is known to be inhibited by CAMP, insulin decreased cAMP concentrations in mammary explants by 70%, and cholera toxin blocked the effects of insulin on poly(ADP-ribose) degradation. This reciprocal relationship between poly(ADP-ribosy1)ation and mammary gland differentiation is further supported by pharmacological studies: in the presence of insulin, cortisol, and prolactin, an inhibitor of the synthetase stimulated a-lactalbumin three-fold over hormone stimulation alone. However, this inhibitor was unable to induce differentiation in the absence of prolactin. Therefore, although there is a close association between a decline in enzyme activity and mammary differentiation, the data are insufficient to support a causal relationship.