We studied the signal pathway through which troglitazone acts in inducing cellular acidosis in LLC-PK1-F⁺ cells in relation to ammoniagenesis and DNA synthesis. Cells were grown to confluent monolayers in 30mm chambers and monitored for pHi by the BCECF assay and activated ERK by phospo-ERK1/2 antibodies. TRO induces a severe cellular acidosis (pHi 6.68± 0.10 vs. 7.28± 0.07 time control at 4minutes, p<0.01) while phospho-ERK1/2 to total ERK 1/2- ratio increases 3.4 fold (p<0.01). To determine whether ERK1/2 was activated by cellular acidosis or TRO was acting via MEK1/2 to activate ERK1/2, cells were pre-treated with specific inhibitors of MEK1/2 activity, PD 098059 and U0126, followed by the addition of TRO or vehicle. With MEK1/2 activity inhibited TRO treatment failed to activate ERK1/2. Preventing ERK1/2 activation abrogated the TRO-induced cellular acidosis and maintained the pHi within the low normal range (7.06± 0.11). To determine whether blocking ERK activation prevents TRO's inhibitory effect on NHE activity, cells were acid-loaded and the recovery response monitored as pHi/ t over a 4minute recovery period. TRO inhibited NHE activity by 85 % (p<0.01) whereas blocking ERK activation restored the response. We measured activated ERK levels and pHi after 3h and 18h exposure to TRO or extracellular acidosis (pHe=6.95) to determine if ERK activation was sustained. Whereas both TRO and extracellular acidosis increased activated ERK and decreased pHi after 3h, only TRO sustained this response at 18h. Furthermore, both enhanced ammoniagenesis and decreased DNA synthesis reflected the effect of TRO to induce and sustain a cellular acidosis.