DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, Mr = 32,000) is a cytosolic neuronal phosphoprotein enriched in dopamine-innervated brain regions which, in its phosphorylated form, acts as an inhibitor of protein phosphatase 1. We have compared the phosphorylation of purified DARPP-32 with that of purified phosphatase inhibitor 1, a widespread inhibitor of protein phosphatase 1. Purified cyclic AMP-dependent protein kinase and cyclic GMP-dependent protein kinase each catalyzed the maximal incorporation of 0.9-1.1 mol of [32P]phosphate/mol of DARPP-32 or phosphatase inhibitor 1, with phosphorylation occurring on threonine residues. Evidence for the existence of a single phosphorylation site in each substrate protein was obtained by two-dimensional thin-layer phosphopeptide mapping of thermolytic digests. Initial rate studies of the phosphorylation of DARPP-32 yielded an apparent Km of 2.4 microM and a kcat of 2.7 S-1 for the catalytic subunit of cyclic AMP-dependent protein kinase, and an apparent Km of 5.4 microM and a kcat of 2.3 S-1 for cyclic GMP-dependent protein kinase. These in vitro results are compatible with a physiological role for the phosphorylation of DARPP-32 by either protein kinase in vivo. Similar studies with phosphatase inhibitor 1 yielded an apparent Km of 5.0 microM and a kcat of 1.4 S-1 for the catalytic subunit of cyclic AMP-dependent protein kinase, and an apparent Km of 25.0 microM and a kcat of 1.2 S-1 for cyclic GMP-dependent protein kinase. A synthetic nonapeptide, corresponding to the phosphorylation site of DARPP-32, was phosphorylated with apparent Km values of 1.12 mM and 1.86 mM and kcat values of 0.22 S-1 and 3.4 S-1 for cyclic AMP-dependent and cyclic GMP-dependent protein kinase, respectively.