Cyclic nucleotide phosphodiesterase activity was studied in 33 malignant neoplastic, 2 benign neoplastic, and 18 nonneoplastic human mammary tissues. Enzyme activity, using both cyclic adenosine 3':5'-monophosphate and cyclic guanosine 3':5'-monosphosphate as substrates, was measured in whole homogenates over a concentration range of 1 to 100 muM. Specific activity was calculated at substrate concentrations of 1 muM (low KM enzyme activity) and 100 muM (high KM activity). Diethylaminoethyl cellulose chromatography was used to separate the different enzyme species. The malignant neoplastic tissues had higher levels of both low-KM cyclic adenosine 3':5'-monophosphate and low-KM cyclic guanosine 3':5'-monophosphate phosphodiesterases. Further, the mean value of the ratio of low-km cyclic adenosine 3':5'-monophosphate to low-KM cyclic guanosine 3':5'-monophosphate activity was higher for the cancer tissues than for the nonneoplastic tissues. Diethylaminoethyl cellulose chromatography indicated the presence of three enzymes in both neoplastic and nonneoplastic mammary tissue. The kinetic as well as regulatory properties of the separated enzymes indicated that they are distinct enzyme activities. The phosphodiesterase properties were similar for neoplastic and nonneoplastic tissues and resembled those described previously in many other mammalian tissues. While both neoplastic and nonneoplastic tissues had detectable levels of the protein activator for phosphodiesterase, the cancer tissues appeared to have a higher level.