Binding of [125I] alpha-rat atrial natriuretic peptide ([125I] alpha-RANP) was measured in glomeruli and pieces of tubule microdissected from rat and rabbit nephrons. High densities of specific ANP binding sites were found only in the glomeruli (10-30 X 10(-18) mol X glom-1), whereas no specific binding could be detected in the proximal tubule, the thin segments of the Henle's loop, the thick ascending limb, the distal tubule and the cortical and outer medullary collecting tubules. Rising the temperature from 4 degrees C to 35 degrees C resulted in biphasic kinetics of binding, suggesting a temperature-dependent inactivation of labelled hormone by glomeruli. At 4 degrees C, specific binding of [125I] alpha-RANP was time and dose-dependent and Scatchard analysis of data indicated an apparent equilibrium dissociation constant of 0.63 nM. Competition experiments revealed the following sequence of stereospecificity for binding to rat glomeruli: RANP 3-28 greater than [125I] alpha-RANP = [125I] alpha-HANP = alpha-RANP = antriopeptin III greater than antriopeptin II, whereas binding was unaffected by pharmacological doses of unrelated peptide hormones, prostaglandins, adrenergic agonists, dopamine, histamine and carbamylcholine. The results indicate that glomerular binding sites might be the physiological ANP receptors.