To investigate the effects of metabolic acidosis on jejunal phosphate and glucose absorption, in vivo and in vitro transport studies were performed on weanling rats fed 1.5% NH4Cl for three days and on group pair-fed controls. Both in vivo and in vitro, acidosis significantly depressed phosphate transport without effecting glucose transport. In vitro, the decrease of phosphate transport was due to a depression of sodium-phosphate cotransport, but not of sodium independent phosphate transport. This corresponded to a significant increase of the Km of sodium-phosphate cotransport with no change of the Vmax. Treatment of the acidotic animals with intraperitoneal 1,25 dihydroxycholecalciferol did not restore phosphate transport to control levels. These studies indicate that in weanling rats, metabolic acidosis selectively suppresses jejunal phosphate transport independent of circulating levels of 1,25 dihydroxycholecalciferol.