Chronic metabolic acidosis increases urine calcium excretion without altering intestinal calcium absorption, suggesting that bone mineral is the source of the additional urinary calcium. In vivo and in vitro studies have shown that metabolic acidosis causes a loss of mineral calcium while buffering the additional hydrogen ions. Previously we have studied changes in femoral, mid-cortical ion concentrations after seven days of in vivo metabolic acidosis induced by oral ammonium chloride. We found that compared to mice drinking only distilled water, the ammonium chloride induced a loss of bone sodium and potassium and a depletion of mineral bicarbonate and phosphate. There is more phosphate than carbonate in neonatal mouse bone. In the current study we utilized a high resolution scanning ion microprobe with secondary ion mass spectroscopy to test that hypothesis chronic acidosis would decrease bulk (cross-sectional) bone phosphate to a greater extent than carbonate by localizing and comparing changes in bone bicarbonate and phosphate after chronic incubation of neonatal mouse calvariae in acidic medium. Calvariae were cultured for a total of 51 hr in medium acidified by a reduction in bicarbonate (HCO₃^-) concentration (pH ~ 7.14, [HCO₃^-] ~ 13) or in control medium (pH ~ 7.45, [HCO₃^-] ~ 26). Compared to incubation in control medium, incubation in acidic medium caused no change in surface phosphate, but a significant fall in cross-section phosphate, with respect to the carbon-carbon bond (C₂)and the carbon-nitrogen bond (CN). Compared to incubation in control medium, incubation in acidic medium caused no change in surface bicarbonate, but a significant fall in cross-section bicarbonate with respect to the C₂ and CN. The fall in cross-section phosphate was significantly greater than the fall in cross-section bicarbonate. The fall in phosphate indicates release of mineral phosphates and the fall in bicarbonate indicates release of mineral bicarbonate, both of which would be expected to buffer the additional protons and help restore the pH toward normal. Thus, a model of chronic acidosis depletes bulk bone proton buffers with phosphate depletion exceeding that of bicarbonate.