The newly discovered peptides extracted from cardiac atria, atrial natriuretic factors (ANFs), when administered parenterally cause renal hemodynamic changes and natriuresis. The nephron sites and cellular mechanism accounting for profound increase in Na+ excretion in response to ANFs are not yet clarified. In the present study we investigated whether synthetic ANF peptide alters the reabsorption of Na+ and reabsorption of solutes cotransported with Na+ in the proximal tubules of rats. Synthetic ANF peptide consisting of 26 amino acids, 4 micrograms/kg body wt/h, or vehicle in controls, was infused to surgically thyroparathyroidectomized anesthetized rats. After determination of the fractional excretion (FE) of electrolytes (Na+, K+, Pi, Ca2+, Mg2+, HCO3), the kidneys were removed and luminal brush border membrane vesicles (BBMVs) were prepared from renal cortex. Solute transport was measured in BBMVs by rapid filtration techniques. Infusion of ANF peptide increased FENa, FEPi, and FEHCO3; but FECa, FEK, and FEMg were not changed. The increase in FENa was significantly correlated, on the one hand, with increase of FEPi (r = 0.9, n = 7; P less than 0.01) and with increase of FEHCO3 (r = 0.89, n = 7; P less than 0.01). On the other hand, FENa did not correlate with FEK, FECa, or with FEMg. The Na+ gradient-dependent uptake of Pi by BBMVs prepared from renal cortex of rats receiving ANF infusion was significantly (P less than 0.05) decreased (-25%), whereas the Na+ gradient-dependent uptake of L-[3H]proline and of D-[3H]glucose or the diffusional uptake of 22Na+ were not changed. ANF-elicited change in FEPi showed a close inverse correlation with decrease of Na+-dependent Pi uptake by BBMVs isolated from infused rats (r = 0.99, n = 7; P less than 0.001). Direct addition of ANF to BBMVs in vitro did not change the Na+ gradient-dependent Pi uptake. In rats infused with ANF, the rate of amiloride-sensitive Na+-H+ exchange across the brush border membrane (BBM) was significantly (P less than 0.05) decreased (-40%), whereas the diffusional 22Na+ uptake (0.5 min) and the equilibrium (120 min) uptake of 22Na+ were not changed. The inhibition of Na+-H+ exchange after ANF was likely due to alteration of the BBM antiporter itself, in that the H+ conductance of BBMVs was not increased. We conclude that synthetic ANF (a) decreases tubular Na+ reabsorption linked to reabsorption of HCO3 in proximal tubules, and (b) inhibits proximal tubular reabsorption of Pi coupled to Na+ reabsorption, independent of secretion and/or action of parathyroid hormone or calcitonin. These ANF effects are associated with inhibition of Na+-Pi synport and of Na+-H+ antiport in luminal BBMs. Our findings document that inhibition of Na+-coupled transport processes in proximal tubules is an integral part of the renal response to ANF.