The sites of action of angiotensin II along the nephron are not well defined and both proximal and distal effects are suggested. Using a microassay that permits measurement of hormone binding in discrete tubule segments, we determined the binding sites of 125I-angiotensin II along the nephron of Sprague-Dawley rats. Specific binding in proximal convoluted tubule (PCT) (at 25 degrees C, pH 7.4) was linearly related to tubule length and saturable, with an apparent maximal binding capacity of approximately 300 amol X cm-1. Binding specificity was verified in competition experiments that revealed significant (P less than 0.001) and comparable competition for radioligand binding by angiotensin II and angiotensin precursor, metabolite, and analogues, whereas unrelated peptides of similar size (bradykinin, ACTH [1-10]) were without effect. The profile of specific angiotensin II binding along the nephron was: PCT, 216 +/- 13; pars recta, 86 +/- 14; medullary thick ascending limb of Henle's loop, 46 +/- 8; cortical thick ascending limb of Henle's loop, 77 +/- 8; distal convoluted tubule, 49 +/- 10; cortical collecting tubule, 15 +/- 1; medullary collecting tubule, 32 +/- 7 amol X cm-1. These results indicate the presence of specific angiotensin II binding sites in all tubule segments studied, but binding capacity was highest in the proximal convoluted tubule, in agreement with transport studies that localize the effects of the hormone in this segment.