We have studied the effects of thrombin (alpha-thrombin) and Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe (SFLL), a peptide agonist of the platelet thrombin receptor in cultured human mesangial cells, and find that SFLL can reproduce the biochemical and morphological effects of thrombin. Treatment of mesangial cells with cAMP-elevating agents causes fragmentation of stress fibers, loss of the vitronectin receptor from sites of focal adhesion, and produces a change in shape from a flat to a more arborized configuration. These effects are prevented by both thrombin and SFLL. Thrombin and SFLL also initiate biochemical signaling events in mesangial cells by stimulating the metabolism of phospholipids. Both thrombin and SFLL stimulate release of inositol phosphates from [3H]inositol-labeled cells, elevation of cytosolic calcium, the formation of [3H]myristic acid-labeled diacylglycerol, an increase in the mass of diacylglycerol, 32P incorporation into phospholipids, and release of unesterified [3H]arachidonic acid from cells prelabeled with [3H]arachidonic acid. When present together, the effects of SFLL and thrombin on diacylglycerol formation, arachidonic acid production, and inositol phosphate production were not additive. This suggested that SFLL and thrombin were acting on the same receptor. This was further supported by our observations that cells pretreated with SFLL and subsequently exposed to thrombin (or vice versa) did not show elevated cytosolic calcium. We also show that phospholipase D is activated by demonstrating production of radiolabeled phosphatidylethanol when cells are treated with SFLL in the presence of ethanol. These findings indicate that SFLL can be used to study the receptor-mediated effects of thrombin in mesangial cells, thereby avoiding thrombin's proteolytic actions.