Cyclic 3',5'-nucleotide phosphodiesterase (PDE) activity in rabbit uterine homogenate was inhibited by indomethacin (10 mug/ml; 66% inhibition) or flufenamic and (10 mug/ml; 60%). Indomethacin (100 mug/ml) reduced uterine prostaglandin E2 (PGE2) content by 80%, but potentiated the stimulatory action of purified cholera toxin (choleragen; 800%) and of exogenous PGE2 (140%) on cyclic AMP accumulation, probably through its inhibitory effect on cyclic AMP destruction. These findings suggest that endogenous PGE2 is not an essential mediator of choleragen action. By contrast, flufenamic acid abolished choleragen and PGE2 action on cyclic AMP production. Unlabeled PGE2 (10 mug/ml), flufenamic acid, indomethacin, and aspirin (100 mug/ml each) inhibited [3H]PGE2 binding to uterine slices by 78, 73, 62, and 20% respectively. It is concluded that while indomethacin and flufenamic acid have similar effects on prostaglandin biosynthesis and PDE activity, only fenamates have an inhibitory effect on the biological action of exogenous PGE2 and choleragen on the stimulation of cyclic AMP production, probably through the inhibition of the binding of PGE2 and choleragen to its specific receptor sites. The diverse biochemical actions of the above drugs indicate that care has to be taken when using these drugs in analyzing the physiopathological roles of prostaglandins.