Rat osteoblasts in culture undergo differentiative changes culminating in the formation of mineralized foci. We here report on the pattern of temporal expression and compartmentalization of osteonectin and of the two small proteoglycans, byglican and decorin. They were constitutively synthesized during in vitro differentiation of rat osteoblasts. The 3 proteins were detected in the conditioned medium and associated with the cell-matrix compartment. Within this compartment they showed prevalent cytoplasmic location and differential distribution on unmineralized noduli was detected for osteonectin and byglican, while decorin was detected throughout the nodules. Along with known functions in the matrix, a possible role in the cytoplasm may have to be sought for these bone cells components.