This article describes a novel technology for quantitative determination of the spatial distribution of CO₃²⁻ substitution in bone mineral using infrared (IR) imaging at ∼6 μm spatial resolution. This novel technology consists of an IR array detector of 64 × 64 elements mapped to a 400 μm × 400 μm spot at the focal plane of an IR microscope. During each scan, a complete IR spectrum is acquired from each element in the array. The variation of any IR parameter across the array may be mapped. In the current study, a linear relationship was observed between the band area or the peak height ratio of the CO₃²⁻ v₃ contour at 1415 cm⁻¹ to the PO₄³⁻ v₁,v₃ contour in a series of synthetic carbonated apatites. The correlation coefficient between the spectroscopically and analytically determined ratios (R² = 0.989) attests to the practical utility of this IR area ratio for determination of bone CO₃²⁻ levels. The relationship forms the basis for the determination of CO₃²⁻ in tissue sections using IR imaging. In four images of trabecular bone the average CO₃²⁻ levels were 5.95 wt% (2298 data points), 6.67% (2040 data points), 6.66% (1176 data points), and 6.73% (2256 data points) with an overall average of 6.38 ± 0.14% (7770 data points). The highest levels of CO₃²⁻ were found at the edge of the trabeculae and immediately adjacent to the Haversian canal. Examination of parameters derived from the phosphate v₁,v₃ contour of the synthetic apatites revealed that the crystallinity/perfection of the hydroxyapatite (HA) crystals was diminished as CO₃²⁻ levels increased. The methodology described will permit evaluation of the spatial distribution of CO₃²⁻ levels in diseased and normal mineralized tissues.