The relationship between kinase C activity and mammary gland differentiation was investigated by following kinase activity throughout the mouse reproductive cycle and by pharmacologically perturbing the kinase, while monitoring biochemical differentiation. Protein kinase C activity declined during pregnancy and remained low throughout lactation, suggesting an inverse relationship with milk protein expression. This negative association was further supported by the use of quercetin (50-100 mumol/l) and gossypol (50 mumol/l), which are both protein kinase C inhibitors. These compounds doubled alpha-lactalbumin levels in mammary explants cultured with hormones. However, a phorbol ester, known to activate protein kinase C, had no effect on alpha-lactalbumin production, although it did stimulate this milk protein 2.5-fold in the presence of the calcium ionophore, A23187. In the absence of raised calcium levels, protein kinase C activity therefore appeared to be inversely correlated with biochemical differentiation; but, in the presence of increased calcium concentrations, both calcium and the kinase acted synergistically to augment hormone-induced alpha-lactalbumin expression.