Bone formation involves several tightly regulated gene expression patterns of bone‐related proteins. To determine the expression patterns of bone‐related proteins during the MC3T3‐E1 osteoblast‐like cell differentiation, we used Northern blotting, enzymatic assay, and histochemistry. We found that the expression patterns of bone‐related proteins were regulated in a temporal manner during the successive developmental stages including proliferation (days 4–10), bone matrix formation/maturation (days 10–16), and mineralization stages (days 16 –30). During the proliferation period (days 4–10), the expression of cell‐cycle related genes such as histone H3 and H4, and ribosomal protein S6 was high. During the bone matrix formation/maturation period (days 10–16), type I collagen expression and biosynthesis, fibronectin, TGF‐β1 and osteonectin expressions were high and maximal around day 16. During this maturation period, we found that the expression patterns of bone matrix proteins were two types: one is the expression pattern of type I collagen and TGF‐β1, which was higher in the maturation period than that in both the proliferation and mineralization periods. The other is the expression pattern of fibronectin and osteonectin, which was higher in the maturation and mineralization periods than in the proliferation period. Alkaline phosphatase activity was high during the early matrix formation/maturation period (day 10) and was followed by a decrease to a level still significantly above the baseline level seen at day 4. During the mineralization period (days 16–30), the number of nodules and the expression of osteocalcin were high. Osteocalcin gene expression was increased up to 28 days. Our results show that the expression patterns of bone‐related proteins are temporally regulated during the MC3T3‐E1 cell differentiation and their regulations are unique compared with other systems. Thus, this cell line provides a useful in vitro system to study the developmental regulation of bone‐related proteins in relation to the different stages during the osteoblast differentiation. © 1996 Wiley‐Liss, Inc.