Two cAMP-activated protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) were purified from bovine thyroid tissue by column chromatography on Sephadex G-200 and DEAE-Sephadex A-25. Maximal stimulation by cAMP of enzyme I was 1.7-fold and of enzyme II was 3.7-fold. The total (basal plus cAMP-activated) specific activities of the purified enzymes I and II were, respectively: 3190 and 6430 protein kinase units (picomoles phosphate transferred per milligram protein per 10 min).The properties of the thyroid protein kinases were found to be similar to those of protein kinases isolated from other tissues. For enzyme I, Km(cAMP) was 20 × 10−8 M, Km(cGMP) was 416 × 10−8 M, and Km(ATP) was 0.02 mM. For enzyme II, Km(cAMP) was 6 × 10−8 M, Km(cGMP) was 250 × 10−8 M, and Km(ATP) was 0.03 mM. For both enzymes, Km(histone) was 1.33 mg/ml, Km(Mg2+) was 2.6 mM, and the pH optimum lay between pH 6.0 and pH 6.5. The cAMP had no effect on the Km values of either I or II for histone, ATP, or Mg2+. The subcellular distribution study indicated that 80% of the total kinase activity was present in the 100 000 × g supernatant fraction.