The effects of acetylcholine and sodium nitroprusside on the activity of c[cyclic]GMP-dependent protein kinase were studied in the perfused rat heart. Acetylcholine produced a dose-dependent increase in cGMP levels and cGMP-dependent rpotein kinase activity and reduced the force of contraction. Both acetylcholine and sodium nitroprusside produced rapid increases in cardiac cGMP, with nitroprusside being the more potent agent. Acetylcholine raised the activity ratio of the cGMP-dependent protein kinase and decreased the force of contraction. Acetylcholine and nitroprusside were slightly additive in their effects on total cGMP levels; the increase in the activity ratio of the cGMP-dependent protein kinase and the decrease in the force of contraction produced by acetylcholine were unchanged by nitroprusside. cGMP produced by acetylcholine, but not nitroprusside, was coupled to protein kinase activation in this tissue.