The proliferative properties and the ability to stimulate the Na⁺/H⁺ antiport activity of a secretory phospholipase A₂ were studied in rat aortic smooth muscle cells in culture. The requirement of the enzymatic activity of phospholipase A₂ to elicit mitogenesis was assessed by the use of ammodytin L, a Ser⁴⁹ phospholipase A₂ from the venom of Vipera ammodytes, devoid of hydrolytic activity. We propose that the proliferative effect is mediated by the same transduction pathway for both proteins. In particular, 1) both secretory phospholipase A₂ and ammodytin L stimulated thymidine incorporation in a dose-dependent manner; 2) both proteins affected the cell cycle, as assessed by cell growth and fluorescence-activated cell sorting experiments; 3) both phospholipase A₂ and ammodytin L increased intracellular pH, a permissive factor for cell proliferation, through activation of the Na⁺/H⁺ antiport; 4) ammodytin L was able to displace the ¹²⁵I-labeled phospholipase A₂ from specific binding sites in a concentration range consistent with that capable of eliciting a cellular response; and 5) the inhibition by heparin was similar for both proteins, taking into account the ratio of heparin to protein. In conclusion, the enzymatic activity of phospholipase A₂ is not required for the stimulation of mitogenesis. The inhibitory effect of heparin combined with its therapeutic potential could help to clarify the role of phospholipase A₂ in the pathogenesis of several preinflammatory situations.