The capacity of cultured renal medullary interstitial cells derived from Dahl salt-sensitive and salt-resistant rats to synthesize prostaglandin E2(PGE2) was compared. Basal and arginine vasopressin (AVP)-induced PGE2production by interstitial cells from salt-resistant rats was fourfold to fivefold higher than corresponding values of those from the salt-sensitive rats. Similarly, basal and AVP-responsive release of [3H]arachidonate were twofold higher by interstitial cells from salt-resistant compared with salt-sensitive rats. Differences in PGE2production were abolished by the calcium inophore A23187 or the addition of exogenous arachidonate. The latter findings suggested a role for altered availability of endogenous arachidonate, possibly mediated by reduced calcium-responsive lipase activity. Basal and AVP-induced increases in cytosolic free calcium concentration, assessed by the aequorin method, were significantly lower in interstitial cells from salt-sensitive versus salt-resistant rats, further supporting a possible role for altered cellular calcium homeostasis. Studies of the potential contribution of various phospholipases and of triglyceride lipase to the release of arachidonate for PGE2synthesis in interstitial cells implicated phospholipase A2activity as a major pathway. When assessed in vitro in cell cytosolic fractions at identical calcium concentration, phospholipase A2activity was lower in interstitial cells from salt-sensitive versus salt-resistant rats. Thus, both reduced cytosolic free calcium and phospholipase A2activity of interstitial cells from salt-sensitive rats may contribute to the diminished capacity of these cells to liberate endogenous arachidonate for PGE2synthesis.