Ammonia and bicarbonate transport by the thick ascending limb of rat kidney was studied to determine whether this segment contributes to the regulation of renal ammonia and net acid excretion. Cortical and medullary thick ascending limbs were perfused in vitro at 1.0-1.5 nl X min-1 X mm-1 with HCO3-buffered solutions. There was no significant net fluid transport. With 4 mM ammonia in bath and perfusate, transepithelial voltage averaged 6-9 mV, lumen positive, and did not differ between the two segments. The mean ammonia concentration in collected tubule fluid was 2.8 mM with cortical segments and 2.3 mM with medullary segments, indicating net absorption of ammonia. Furosemide (10(-4) M) in the perfusate eliminated ammonia absorption in medullary thick ascending limbs and converted net absorption to net secretion in cortical thick ascending limbs. Furosemide reduced transepithelial voltage to near zero in every tubule. Cortical and medullary thick ascending limbs also absorbed bicarbonate, indicating that their tubule fluid was acidified relative to the bath. Therefore, absorption of ammonia could not have occurred by nonionic diffusion. The absorption most likely was due to direct transport of NH4+. The possible mechanisms involved are discussed, and it is proposed that absorption of ammonia by thick ascending limbs provides a source for its accumulation in the renal medulla and secretion into the collecting ducts.