Mesangial cells respond to vasopressin by contraction and increased prostaglandin production. The purpose of the present study is to characterize vasopressin receptors from these cells. Glomeruli were isolated from rat kidneys and plated for explant growth of mesangial cells. Membranes were prepared from cells grown for 6 wk and tested for their ability to bind [3H]vasopressin (lysine vasopressin). These membranes contained a single class of specific vasopressin binding sites [equilibrium dissociation constant (Kd) = 10 +/- 1 nM, maximal binding capacity (Bmax) = 270 +/- 7 fmol/mg protein for 5 determinations]. Vasopressin induced a dose-dependent (apparent Kact value = 2 nM) accumulation of labeled inositol phosphates in myo[3H]inositol-prelabeled mesangial cells incubated in the presence of 10 mM of Li. Conversely, vasopressin failed to alter the adenylate cyclase activity of mesangial cell membranes. Competition experiments with a series of vasopressin structural analogues that have different degrees of affinity for V2-(renal), V1a- (vascular and hepatic), and V1b- (adenohypophyseal) receptors, indicated that vasopressin receptors from rat glomerular mesangial cells resemble the V1a- receptor subtype.