A new assay for cyclic nucleotide phosphodiesterase has been developed by using reverse-phase column chromatography for the separation of product and substrate of the enzymatic reaction. The polar 5'-nucleotides are not retarded by the column, while the more lipophilic cyclic nucleotides bind to the column. Properties such as pH and ionic strength of the incubation mixture or the elution buffer have only minor effects on the elution pattern. The assay by reverse-phase chromatography has several advantages above other assay methods currently employed; it is fast and simple, has a very low blank (0.2%), and is very sensitive (1 fmol). The assay can be used for different substrates (cyclic AMP, cyclic GMP, cyclic IMP) without modification of the conditions. The usefulness of the assay is demonstrated by transient kinetic measurements on a time scale in seconds of a cGMP-dependent cGMP-specific phosphodiesterase from the cellular slime mold Dictyostelium discoideum.