To investigate the effect of reduction in renal medulla osmolality on loop of Henle (LOH) net bicarbonate reabsorption, clearance and microperfusion experiments were performed on Sprague-Dawley rats. The decrease of renal medulla osmolality was induced by intravenous infusion of either a large dose of mannitol (mannitol protocol) or a hypotonic solution (hypotonic protocol) delivered at a rate to match the sodium and bicarbonate load of the control period. During the mannitol protocol, clearance data demonstrated a rise in glomerular filtration rate (GFR), renal plasma flow, urine pH, and fractional bicarbonate excretion. On the contrary, microperfusion experiments, performed in the absence of mannitol in the tubular perfusate, revealed a significant increase both in the absolute and fractional LOH bicarbonate transport. During the hypotonic protocol, there was a decrease in GFR, associated with an increase in fractional excretion of bicarbonate. In the microperfusion experiments, hypotonic saline, similar to mannitol, stimulated absolute and fractional LOH bicarbonate transport. Net reabsorption of chloride, measured under the same experimental conditions, was also found to be activated. Therefore, the intravenous infusion of hypotonic solution affected the LOH transepithelial net reabsorption of both bicarbonate and chloride. We hypothesize that the increase in the transport rate of these two anions, along the same segment and in similar experimental conditions, may be mediated, at least in part, by decreased medullary tonicity, which is one factor common both to hypertonic mannitol and hypotonic saline infusion.