We studied the proton secretion mechanisms involved with pH_i regulation in IRPTC cells, a SV40-immortalized cell line derived from rat proximal tubule, and characterized the effects of serum deprivation thereupon. Using pH_i measurements with the fluorescent probe BCECF, we demonstrated that the IRPTC cells express both Na⁺/H⁺ exchanger and H⁺- ATPase, but only NHE1 is modulated by serum deprivation. In these cells, 24h of serum starvation increased pH_i [from 7.08 ± 0.008(n=34) to 7.18 ± 0.018(n=33)] as well as the pH recovery rate from intracellular acidification with NH₄Cl [from 0.29 ± 0.022 pH units/min(n=14) to 0.50 ± 0.024 pH units/min(n=14)], without modifying their buffering capacity. These effects were followed by several modifications in morphological features, indicating an increase in differentiation status. The altered activity of NHE1 was consistent with an increase of both transcription and translation of the antiporter, since the utilization of actinomycin D and cycloheximide significantly inhibited the up-regulation of NHE1 induced by serum withdrawal. Inhibition of tyrosine phosphorylation by genistein blocked the serum deprivation-dependent activation of NHE. Moreover, the pharmacological inhibition of MEK1/2, the upstream activator of ERK1/2 by UO126 significantly inhibited the stimulatory effect of serum starvation on Na⁺/H⁺ exchanger activity, while the putative p38 MAPK inhibitor SB 203580 failed to cause any effect on pHi recovery rates. Our findings indicate that during IRPTC cell differentiation by serum deprivation, there was a net enhancement of NHE1 activity. This up-regulation of NHE by serum removal was consistent with an increase of RNA and protein synthesis of the exchanger, which depends on tyrosine kinase phosphorylation and ERK pathway activation.