Cyclic nucleotide phosphodiesterase activity (3':5'-cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17), which is activatable by Ca(2+)-dependent regulator protein (CDR), has been identified in cycled microtubule preparations from bovine brain. By using various methods to fractionate the microtubule preparation into subfractions (e.g., phosphocellulose chromatography to obtain purified 6S tubulin and soluble microtubule-associated proteins, and gel exclusion chromatography on Bio-Gel A-150m to obtain 10-nm filaments), we found that all the fractions exhibited some enzymic activity, but that most of the phosphodiesterase activity was localized in the 10-nm filament fraction. By using cyclic GMP as substrate, a specific activity of 921 +/- 168 pmol/mg of filament protein.min was determined. Also, 10-nm filaments were prepared directly from brain homogenates by differential centrifugation and gel exclusion chromatography. This fraction also contained phosphodiesterase activity but of slightly lower specific activity (752 +/- 9 pmol/mg of protein.min). The filament-associated enzymic activity was stable during storage (-70 degrees C) and to several salt extractions at moderate ionic strength (0.5 M); the latter finding indicates that the phosphodiesterase is not adsorbed to the filaments via nonspecific electrostatic interactions. Although a chelating agent was present in the initial homogenization buffer and generally in all buffers used in preparing fractions, an activator of a smooth muscle phosphodiesterase was released upon boiling the 10-nm filaments. This activator obtained in the boiled supernatant was Ca(2+)-sensitive, trifluoperazine-sensitive, and stimulated smooth muscle phosphodiesterase to nearly the same extent as purified (exogenous) CDR; thus, it probably represents filament-associated CDR.