We present electron microscopic and electron spectroscopic images of putative nucleation sites and early mineral deposits during intramembranous ossification of the murine perichondrial ring. Electron spectroscopic imaging (ESI) permits the quantitative determination and direct visualization of spatial distribution of atomic elements within specimens at high spatial resolution. In this study ESI was used to determine the elemental distributions of phosphorus, sulfur, and calcium. Nucleation and subsequent mineralization in the perichondrial ring occurred sequentially along the longitudinal axis. Proximal regions of the ring contained a matrix with only a few nucleation sites that are characterized in conventional electron micrographs as small loci of low-density material in which dense particles are located. Elemental maps of these sites that we obtained by ESI reveal a sulfur-containing matrix in which localized concentrations of phosphorus occur. With further maturation the loci became centers for the genesis of numerous dense rods or crystals. These mineral deposits contained increased concentrations of P, S, and Ca, compared with the surrounding matrix. The appearance of S at nucleation sites and its persistence in developing mineral deposits suggests that a sulfur-containing moiety may serve as a locus within the osteoid matrix to attain high local concentrations of Ca and P, which leads to the controlled local formation of calcium phosphates. Calcification of the perichondrial ring has been found to occur in the absence of matrix vesicles, which illustrates that these membrane-bounded organelles are not obligatory sites for nucleation in this matrix.