Abstract Cyclic nucleotide phosphodiesterase (EC 3.1.4.17) activity was characterized in crude and (NH4)2SO4-fractionated extracts of the green alga Chlamydomonas reinhardtii. With cyclic AMP as substrate, the formation of 5′-AMP was optimal at pH 8.5 and required the presence of a sulfhydryl reagent and a divalent cation, Mg2+ and Mn2+ being the most effective. Methylxanthines, papaverine, inorganic phosphate and pyrophosphate, ATP and Co2+ inhibit the enzyme, while imidazole stimulates the activity. At 2 mM substrate concentration, cyclic CMP is hydrolyzed at three times the rate for cyclic AMP, while the cyclic 3′,5′-derivatives of GMP, IMP, TMP and UMP were hydrolyzed at lower rates. No hydrolysis of N 6 ,O 2′ - dibutyryl cyclic AMP was observed. The Chlamydomonas reinhardtii enzyme closely resembles cyclic nucleotide phosphodiesterases from other micro-organisms and animal tissues, and is distinctly different from cyclic nucleotide phosphodiesterases that have been found in higher plants.