Intracellular pH regulation of osteoblasts is of a great importance in the process of bone formation and resorption, and has been suggested to be mediated via intracellular Ca²⁺ and cAMP messenger systems. To elucidate the mechanism of modulation of intracellular pH by parathyroid hormone and PMA(Phorbo1-12-myristate-13-acetate), effects of these agonists on the individual transporter system, Na⁺-H⁺ antiporter and Cl⁻-HCO₃-(−OH⁻) exchanger, were investigated. Intracellular pH and Ca²⁺ were measured by using the fluorescent dye BCECF and fura-2, respectively, in UMR-106 cell monolayer grown on glass coverslip. Addition of tumor promotor, PMA(luM) caused 0.14 unit pH rise of resting intracellular pH(pHi) and 38% increase of the initial rate of pHi recovery after cytosolic acid load. Perfusion of Cl⁻-free solution resulted in rapid cytosolic alkalinization of which the rate was increased 26% by preincubation of PMA. Ca²⁺ ionophore, ionomycin (1uM) decreased resting pHi by 0.17 unit, but had no effect on the initial rate of pHi recovery after cytosolic acid load. However, the addition of ionomycin augmented the initial rate of pHi increase after Cl⁻-depletion outside the cells by 34% over the control. Stimulation of cells with parathyroid hormone(10-8M) caused an initial acidification (0.27 unit) followed by cytosolic alkalinization, with inhibiting effect on the initial rate of pHi recovery after acid load (42%). But parathyroid hormone did not have any significant effect on the rate of pHi increase after Cl⁻-depletion. PMA caused a sustained increase of intracellular Ca²⁺, of which the peak depended on the concentration of Ca²⁺ in extracellular medium. Ionomycin caused a transient increase of Ca²⁺ but PTH had no significant increase of intracellular Ca²⁺ in the concentration range of 10-6M to 10-12M tested. 10-8M PTH increased cAMP levels by about 10-fold and 10-10M PTH did by 1.6-fold. PMA, which increased cytosolic Ca²⁺ concentration, also had an stimulatory effect on cAMP production in the concentration range of 10-5M to 10-6M by 2-fold. These findings suggest that in UMR-106 cells Ca²⁺ and cAMP can influence pHi by altering the activity of pHi regulatory transporter system, and parathyroid hormones modulate pHi by inhibiting Na⁺-H⁺ antiporter via intracellular increase of cAMP, which is probably accounts for the inhibitory effect of parathyroid hormone on the proliferation of osteoblasts.