The Cgamma and Calpha subunits of the cAMP-dependent protein kinase (PKA) contain 350 amino acids that are highly homologous (83% amino acid sequence), with 91% homology within the catalytic domain (a.a. 40-300). Unlike Cgamma, the Calpha subunit has been readily purified and characterized as a recombinant protein in vitro, in intact cells, and in vivo. This report describes for the first time the expression, purification, and characterization of Cgamma. The expression of active Cgamma was eukaryote-specific, from mammalian and insect cells, but not bacteria. Active recombinant Cgamma was optimally expressed and purified to homogeneity from Sf9 cells with a 273-fold increase in specific activity and a 21% recovery after sequential CM-Sepharose and Sephacryl S-300 chromatography. The specific activity of pure Cgamma was 0.31 and 0.81 U/mg with kemptide and histone as substrates, respectively. Physical characterization showed Cgamma had a lower apparent molecular weight and Stokes radii than Calpha, suggesting differences in tertiary structures. Steady-state kinetics demonstrated that like Calpha and Cbeta, Cgamma phosphorylates substrates requiring basic amino acids at P-3 and P-2. However, Cgamma generally exhibited a lower Km and Vmax than Calpha for peptide substrates tested. Cgamma also exhibited a distinct pseudosubstrate specificity showing inhibition by homogeneous preparations of RIalpha and RIIalpha-subunits, but not by pure recombinant protein kinase inhibitors PKIalpha and PKIbeta, PKA-specific inhibitors. These studies suggest that Cgamma and Calpha exhibit differences in structure and function in vitro, supporting the hypothesis that functionally different C-subunit isozymes could diversify and/or fine-tune cAMP signal transduction downstream of PKA activation.