Because of the inaccessibility of the thick ascending limb (TAL) to micropuncture, it has been difficult to evaluate the effects of ADH and atrial natriuretic factor (ANF) on TAL salt transport in vivo. The purpose of the present study is to assess the effects of these hormones on TAL NaCl reabsorption in vivo. Although passive NaCl permeability and unidirectional Na fluxes were not measured, the microstop-flow conductivity technique that was used in these studies controls solute delivery, permits the measurement of the rate of net NaCl reabsorption, and tests the capacity of the TAL to establish a limiting gradient. Despite the presence of a dramatic natriuretic response, a natural extract of ANF did not significantly increase the minimum NaCl concentration reached by the TAL after 60 s of stop-flow. However, 1-[desamino]-8-D-arginine vasopressin (dDAVP) increased by 23%, the rate at which luminal Na concentration declined, and reduced by 28% the minimum NaCl concentration achieved by the TAL. Therefore, these results do not demonstrate a significant direct peritubular effect of ANF on TAL NaCl permeability in outer cortical nephrons, but support a role for antidiuretic hormone in enhancing TAL NaCl reabsorption in vivo.