Angiotensin II (Ang II) acting through its type 1 (AT1) receptor stimulates total ammonia (tNH₃) production by the proximal tubule. The present studies explored the role of Ang II type 2 (AT2) receptors in modulating the stimulatory effects of Ang II on tNH₃ production. Mouse S2 proximal tubule segments derived from 18-h and 7-d acid loaded mice, and non-acid-loaded controls were dissected and microperfused in vitro. Adding Ang II to the luminal perfusion solution resulted in different increments in tNH₃ production rates in tubules derived from 18-h versus 7-d acid-loaded mice such that the increase in tNH₃ production with Ang II was higher in tubules derived from 18-h acid-loaded mice compared to those derived from control and 7-d acid-loaded mice. Adding the AT2 receptor blocker PD123319 with Ang II increased Ang II-stimulated tNH₃ production in S2 segments from control and 7-d acid-loaded mice but not in those from 18-h acid-loaded mice and this increased effect of PD123319 was associated with higher AT2 receptor protein levels in brush border membranes. Studies on cultured proximal tubule cells demonstrated that 2-h exposure to pH 7.0 reduced the modulating effect of PD123319 on Ang II-simulated tNH₃ production and reduced cell surface AT2 receptor levels. We concluded that AT2 receptors reduce the stimulatory effect of Ang II on proximal tubule tNH₃ production and that the time-dependent impact of AT2 receptor blockade on the Ang II-stimulated tNH₃ production corresponded to time-dependent changes in AT2 receptor cell surface expression in the proximal tubule.