The catalytic subunits of cAMP-dependent protein kinases I and II were isolated from rabbit skeletal muscles in a homogeneous state. The specific phosphotransferase activities of homogeneous preparations of catalytic subunits were 8 mumol/mg X min (type I) and 6 mumol/mg X min (type II). In order to elucidate the mechanisms of the phosphotransferase reaction, the steady-state kinetics method and an inhibitory analysis involving the phosphotransferase reaction products, ADP and phosphohistone H1, were used. It was shown that phosphorylation of histone H1 catalyzed both by protein kinases I and II occurs via a random "bi-bi" mechanism. The values of constants for kinetic equation of the phosphotransferase reaction coincide with those for the catalytic subunits of both protein kinase types and are equal to 11 microM (KmATP), 60 microM (KmH1), 5.0 microM (KSATP) and 27 microM (KSH1). The value of the competitive inhibition constant for Mg-ADP (KiADP) is also identical for the catalytic subunits of types I and II and is equal to 30 microM. In both cases, the phosphorylated histone H1 inhibits the phosphotransferase reaction; this inhibition is partly competitive with respect to histone H1.