Cyclic nucleotide phosphodiesterase activities in human neutrophils were characterized. Neutrophil sonicates exhibited high-affinity and low-affinity cAMP phosphodiesterase activities, with apparent Km values of 1.9 microM and 112 microM, respectively. No cGMP phosphodiesterase activity was detected. Approx. 70% of cAMP phosphodiesterase activity measured at 1 microM cAMP was present in the soluble subcellular fraction, and the remainder was localized in the particulate fraction. Chromatography of the soluble subcellular fraction on DE-52 ion-exchange resin yielded a low-affinity cAMP phosphodiesterase activity and a high-affinity cAMP phosphodiesterase activity. The soluble high-affinity cAMP phosphodiesterase activity exhibited moderate calmodulin sensitivity. After incubation of intact neutrophils with N-formylmethionylleucylphenylalanine (fMet-Leu-Phe), a 25-30% increase in the activity of the high-affinity cAMP phosphodiesterase activity was observed in the sonicate and in the soluble fraction. Maximal increases were achieved after 2 min of incubation and the increases persisted for at least 10 min. The increase in activity was independent of calmodulin and guanine nucleotide regulatory proteins. These results indicate that a soluble high-affinity cAMP phosphodiesterase comprises the majority of phosphodiesterase activity in neutrophils and that increases in this activity may contribute to the regulation of cAMP levels in neutrophils during activation.