The regulation of type I cAMP-dependent protein kinase during the differentiation of L6 myoblasts has been investigated in order to assess a possible role for this enzyme in the control of myogenesis. Immunoblot analysis showed that the levels of the type I cAMP-dependent protein kinase regulatory subunit (RI) increased 3-fold during differentiation. However, measurement of RI mRNA levels using an RI cDNA probe showed that this increase was not regulated transcriptionally. Determinations of the turnover rate of RI demonstrated that the subunit becomes 3-fold more stable in the differentiated cells. Therefore, it appears that the increase is due to a decrease in the rate of degradation of RI. The increase in both the amount and stability of RI could be reversed by treating myotubes with cAMP analogues or forskolin. Furthermore, during differentiation there was a large decrease in cAMP levels. It was, therefore, concluded that the increase in RI levels seen during differentiation is probably due to an increase in the stability of the subunit as a result of a decrease in intracellular cAMP concentrations.