Abstract 1. 1. N6,O2′-Dibutyryl AMP, N6-monobutyryl cyclic AMP and O2′-monobutyryl cyclic AMP and O2′-monobutyryl cyclic AMP are able to activate to the same maximal velocity purified mucle cyclic AMP-dependent protein kinase. The concentrations required for this maximal stimulation, however, are widely different, the most effective being the N6-monobutyryl derivative. 2. 2. The relatively low Ka for N6-monobutyryl cyclic AMP (2·10−7–3·10−7 M), Ka for cyclic AMP 8·10−8 M), the observed similarity wich cyclic AMP in its ability to dissociate the protein kinase, and the reported formation of N6-monobutyryl cyclic AMP from dibutyryl cyclic AMP in many cells strongly suggests a direct effect of the N6 derivative on the protein kinase, not involving formation of free cyclic AMP or inhibition of phosphodiesterase.