Osteoporosis is a skeletal disorder that is characterized by the loss of bone mineral density (BMD) resulting in increased risk of fracture. However, it has been shown that BMD is not the only indicator of fracture risk, as the strength of bone depends on a number of factors, including bone mass, architecture and material properties. Physiological mineral deposition requires the formation of a properly developed extracellular matrix (ECM), which recruits calcium and phosphate ions into the synthesis of apatite crystals. Temporal and spatial compositional and structural changes of biological apatite greatly depend on the properties of the crystals initially formed. As such, Fourier-transform infrared microspectroscopy (FTIRM) is capable of examining adaptive remodeling by providing compositional information such as the level of mineralization and carbonate substitution, as well as quality and perfection of the mineral phase. The objective of this study was to evaluate the in vitro mineralization development of MC3T3-E1 murine calvarial preosteoblasts cultured on different substrata by comparing FTIRM measurements from two subclones (mineralizing subclone 4 and nonmineralizing subclone 24) maintained in culture for up to 21 days. The results showed that modulation of the substrate surface using a thin coating of sulfonated polystyrene (SPS) provided favorable conditions for the development of a mineralizable ECM and that the mineral formed by the osteoblasts was similar to that of fully mineralized bone tissue. Specifically, the mineralizing subclone produced significantly more mineral phosphate when cultured on SPS-coated substrates for 21 days, compared to the same culture on bare substrates. In contrast, the level of mineralization in nonmineralizing subclone was low on both SPS-coated and uncoated substrates. The mineralizing subclone also produced comparable amounts of collagen on both substrates; however, mineralization was significantly higher in the SPS culture. The nonmineralizing subclone produced comparable amounts of collagen on day 1 but much less on day 21. Collagen maturity ratio increased in the mineralizing subclone from day 1 to day 21, but remained unchanged in the nonmineralizing subclone. These results suggest that SPS-treatment of the substrate surface may alter collagen remodeling; however, other factors may also influence osteoblast mineralization in the long term.