In this study, we examined the role of insulin, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) cascade in activation of protein phosphatase-1 (PP-1) by using three complementary approaches. First, differentiated L6 cells were acutely exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA, 400 nM) to activate PKC. In these cells, TPA caused 32% stimulation of PP-1 activity. The PP-1 stimulation by TPA was comparable to stimulation by insulin (t1/2 = 1 min and EC50 = 5 nM) with a maximum effect in 5 min. The effects of insulin and TPA were not additive. Insulin and TPA also stimulated MAPK (> 2-fold increase over basal, with myelin basic protein as a substrate). ML-9, a myosin light chain kinase inhibitor, blocked the effects of insulin and TPA on both MAPK and PP-1 activation. In the second approach, PKC was down-regulated by chronic treatment with TPA. In these cells subsequent effects of insulin on MAPK and PP-1 activation were blocked, without an effect on basal enzyme levels. In the third approach, two selective inhibitors of PKC, calphostin and chelerythrine chloride, were used to inhibit PKC. These inhibitors completely prevented insulin and TPA stimulation of MAPK and PP-1 and blocked insulin-induced translocation of PKC to the plasma membranes. We conclude that PKC plays an important role in insulin stimulation of PP-1 via the activation of MAPK cascade.