125I-Angiotensin (Ang) IV and (125)I-divalinal Ang IV [AT receptor subtype 4 (AT(4))] receptor agonist and putative antagonist, respectively] were used to characterize the AT(4) receptor in Mardin-Darby bovine kidney epithelial cells (MDBK cell line). Both (125)I-Ang IV and (125)I-divalinal Ang IV bound to a single high-affinity site (K(D) = 1.37 and 1.01 nM, respectively) and to a comparable density of binding sites (B(max) = 1335 and 1407 fmol/mg protein, respectively). Competition of either radiolabeled ligand with several Ang related peptides demonstrated similar displacement affinities in the following affinity order: Ang IV = divalinal Ang IV > Ang III > Ang II > losartan = PD 123177. Guanosine-5'-O-(3-thio)triphosphate or sulfhydryl reducing agents did not affect the binding of either radiolabeled ligand. Brief exposure of MDBK cells to Ang IV or divalinal Ang IV (0.1 nM to 1 microM) caused a concentration-dependent rise in intracellular calcium concentration levels with a reduced calcium response observed with Ang IV at micromolar concentrations. These results indicate that Ang IV and divalinal Ang IV bind with high affinity to the same receptor and that the MDBK AT(4) receptor is not coupled to a classic G protein, nor are sulfhydryl bonds important in regulation of receptor affinity. The MDBK AT(4) receptor appears to be pharmacologically similar to that described in nonrenal tissues. Functional studies suggest that AT(4) receptor activation can increase intracellular calcium concentration levels in MDBK cells and that divalinal Ang IV possesses agonist activity with respect to this particular intracellular signaling system.