The heterogeneous nature of renal ammonia production and transport necessitated the adaptation of the in vitro microperfusion technique to study ammonia production and transport by specific segments of the proximal tubule. Specific proximal tubule segments were dissected from mouse kidneys and microperfused in vitro with physiologic buffer solutions. This approach has demonstrated the important effects of luminal perfusion and luminal flow rate, in vitro and in vivo metabolic acidosis and in vitro potassium concentration on total ammonia production rates in proximal tubule segments. Studies examining net luminal ammonia secretion have suggested an important role of the Na(+)-H+ exchanger in the mechanism of ammonia secretion by the proximal tubule.