Most of the cyclic nucleotide phosphodiesterase (PDE) activity of pregnant human myometrium was found in the soluble fraction. Chromatography of this fraction on DEAE-cellulose resolved two peaks of PDE activities. Peak I hydrolyzed both cAMP and cGMP and was activated by the Ca2+-calmodulin complex. Peak II, insensitive to this complex, hydrolyzed specifically cAMP. Sucrose gradient centrifugation of the soluble fraction resolved three peaks (A, B, C) of cAMP PDE activities, and only the first two peaks (A, B) were active towards cGMP. A subsequent sucrose gradient centrifugation of peak I, previously determined by DEAE-cellulose, allowed us to restore two peaks A and B identical to those directly obtained from the soluble fraction: peak A hydrolyzes both substrates, while peak B is specific to cGMP hydrolysis. For peak II, a single large cAMP PDE activity peak is generated. Considering Ca2+ and cAMP as intracellular messengers in the control of uterine motility, the characterization of the different forms of PDE in pregnant human myometrium will be of importance in developing improved tocolytic therapy.