In vitro metabolic acidosis (Met) induces greater bone mineral resorption than respiratory acidosis (Resp). Met, but not Resp, inhibits osteoblasts which control many aspects of osteoclastic function. To determine whether at a similar decrement in extracellular pH, Met and Resp would induce different changes in intracellular pH (pH(i)) and/or intracellular calcium concentration ([Ca2+](i)) of osteoblasts, we measured pH(i) and [Ca2+](i) in an osteoblast-like rat osteosarcoma cell line (UMR-106). Cells were grown to confluence on glass slides and loaded with either 1.5 μM BCECF, for pH(i), or 1.5 μM Fura-2, for [Ca2+](i), in control (Ctl; pH ~ 7.40, P(Co2) ~ 40, [HCO3-] ~ 24) medium. The fluorescence ratio at excitation wavelengths of 502 and 440 nm was measured for pH(i) and at 340 and 380 nm for [Ca2+](i). Following a baseline scan in Ctl medium, cells were transferred to either Met (pH ~ 7.10, P(Co2) ~ 40, [HCO3-] ~12), Resp (pH ~ 7.10, P(Co2) ~ 80, [HCO3-] ~ 24) or Ctl conditions. Medium pH, P(Co2) and [HCO3-] were held constant over the course of the experiment. Compared to Ctl, pH(i) was lower in Met (P < 0.001) and even lower in Resp (P < 0.001 vs. Met and vs. Ctl). These changes were maintained over the period of observation. Compared to Ctl, [Ca2+](i) was higher in Met (P < 0.001) and even higher in Resp (P < 0.001 vs. Met and vs. Ctl) within 20 to 100 seconds. However, after 100 seconds [Ca2+](i) was not different in the three groups. During chronic 24 and 48 hour incubations there was no change in pH(i) or [Ca2+](i) with Met compared to Ctl; however, there was a marked decline in pH(i) with Resp compared to Met and Ctl and in [Ca2+](i) with Resp compared to Met. Thus at a similar decrement in medium pH, Met and Resp induce a differential response of osteoblastic pH(i) and [Ca2+](i); how this relates to differences in bone resorption remains to be determined.