This study was aimed at identifying the renal sites of the antinatriuretic action of insulin by evaluating whether this hormone may alter the function of Na-K-ATPase in specific nephron segments. For this purpose, possible actions of insulin on the rate of 86Rb uptake were evaluated in vitro on single segments of proximal convoluted tubule (PCT), thick ascending limb, and collecting tubule microdissected from collagenase-treated kidneys of normal rats. Results indicate that physiological concentrations of insulin inhibited by 44% the initial rate of ouabain-sensitive 86Rb uptake in the medullary and cortical thick ascending limb, whereas it increased it by 40% in proximal tubules and by 60% in both cortical and medullary collecting tubules. The kinetics and dose dependence of insulin actions were different in the thick ascending limb, the PCT, and the collecting tubule, with the latter less sensitive but displaying an earlier response to insulin than the PCT and the thick ascending limb.