Phosphatidic acid (PA) has been previously shown to activate specifically some of the isoforms of type 4 cylic nucleotide phosphodiesterases (PDE-4) in an acellular system. In the present work, we have investigated the mechanism of PA-activating effect by using a recombinant PA-sensitive isoform, PDE-4D3. The enzyme was specifically activated by acidic phospholipids, but not by zwitterionic phospholipids or anionic detergents. The importance of the role of PA acidic groups in the activation process was confirmed by studying the influence of pH and ionic strength on activation. Crosslinking experiments suggested that PA might influence the ability of PDE-4D3 to form dimers. Binding studies performed with radiolabeled PA showed that PA binds to a PDE-4D3 preparation in a saturable manner. Specifically bound PA was displaced by anionic, but not by zwitterionic phospholipids. With a preparation of PDE-4B2, a PDE-4 isoform insensitive to PA activation, PA binding was only displaced by high concentrations of unlabeled PA, suggesting that high-affinity PA binding sites are only present on PDE-4D3. These data support the hypothesis that PA-activating effect depends on direct binding of the effector on specific sites carried by the PDE-4D3 protein.