A simple yet precise microassay for as little as 5 ppmoles of 3 ',5 '-cyclic adenosine monophosphate or 3 ',5 '-cyclic guanosine monophosphate has been developed through a modification of the isotope dilution principle. The hydrolysis of tritium-labeled cyclic nucleotides by a uniform phosphodiesterase reaction is diminished by the addition of nonlabeled cyclic nucleotides. Cyclic nucleotides are isolated from tissue samples by perchloric acid extraction, absorption and elution from small anion-exchange columns, and lyophilization. A simple preparation from rat brain contains both 3 ',5 '-cyclic adenosine monophosphate and 3 ',5 '-cyclic guanosine monophosphate phosphodiesterase activities. Enzymz reaction mixtures include snake venom to provide an excess of 5 '-nucleotidase activity. The nucleosides produced in the coupled enzyme system are separated from unreacted substrate by treatment

The material presented is taken in part from the thesis submitted in 1962 to the Graduate Faculty of the University of Washington in partial fulfillment of the requirements for the Ph.D. degree.

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