A method has been developed for counting active sites of cyclic-AMP-dependent protein kinase. Known concentrations of a synthetic peptide similar to a fragment of the endogenous inhibitor of the kinase were included in otherwise routine assay mixes containing several different volumes of enzyme stock solution. The concentration of active sites of the catalytic subunit of the cyclic AMP-dependent protein kinase in the stock solution was then determined by fitting observed velocities to an equation that accounts for the presence of a tight-binding inhibitor. The method yielded estimates of catalytic subunit concentration comparable with those derived from more traditional measures of catalytic subunit concentration. Both purified and heterogeneous samples were assayed, since active-sites counting assumes only a mutually specific, high-affinity interaction between enzyme and inhibitor and does not require that samples be pure. In principle, the method can be adapted to other protein kinases for which a specific, tight-binding, reversible inhibitor is available.