Fourier transform infrared spectroscopy (FTIR) was used to characterize the organic and mineral phases present during the induction of mineral formation by collagenase-released matrix vesicles (CRMV) during incubation in a synthetic cartilage lymph in vitro. CRMV mineralization, which occurs in the absence of alkaline phosphatase organic phosphate substrates, is characterized by an initial short lag period of limited Ca2+ accumulation, followed by a period of rapid Ca2+ uptake, and finally, by a plateau period during which Ca2+ accumulation continued at a slower rate. FTIR spectra taken at timed intervals during the induction of mineralization revealed the presence of absorptions characteristic of protein, phospholipid, and mineral components in the CRMV. These became progressively more intense with time. To reveal underlying changes occurring during the successive stages of Ca2+ accumulation, FTIR spectra of nascent (or demineralized) CRMV were computer-subtracted from subsequent spectra, nulling on the C-H stretch modes characteristic of the lipid acyl chains. These difference spectra showed little change during early Ca2+ loading, revealing that mineral ions initially accumulated in a form similar to that present in nascent matrix vesicles (MV). During the period of rapid Ca2+ uptake prior to appearance of crystalline mineral, difference spectra revealed subtle changes in the carbonyl and amide nitrogen stretch modes indicative of protein conformational changes. The first definable mineral phase appeared late in the rapid Ca2+ uptake period and was a distinct, crystalline octacalcium phosphate (OCP)-like phase. With time, the OCP-like precursor became more apatitic in character. There was no evidence that any amorphous calcium phosphate phase formed during the MV mineralization sequence. The mature MV mineral phase closely resembled hydroxyapatite formed via an OCP precursor and was similar to other biological apatites that show a substantial incorporation of carbonate.