To assess the presence and nature of steady-state anion current across the basolateral membrane in in vitro rabbit proximal convoluted tubules bathed and perfused with a high-chloride, low-bicarbonate solution simulating late proximal tubular fluid, steady-state basolateral cell membrane potential difference (Vb1) was measured by conventional microelectrodes. The mean value of Vb1 was -52 mV. Addition of 1 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) to the bath solution hyperpolarized Vb1 by 30 mV, suggesting the presence of basolateral anion current. Total chloride removal did not change Vb1 significantly, and formate, a presumptive stimulant of electroneutral sodium chloride transport, depolarized Vb1 both in the presence and absence of chloride, suggesting that the formate-stimulated change in Vb1 was chloride independent. In the total absence of chloride and bicarbonate, 1 mM bath SITS and 0.1 mM lumen and bath acetazolamide hyperpolarized Vb1 by 27-35 and 23 mV, respectively. These results suggest that the SITS-sensitive change in Vb1 is independent of chloride and associated with a basolateral anion current that is predominantly due to bicarbonate exit. In the absence of exogenous CO2, cell-to-bath HCO3-dependent anion current can be derived from metabolic CO2.