The in vivo expression of fibronectin, type I collagen, and several non-collagenous proteins was correlated with the development of bone in fetal and early neonatal rat calvariae. Fibronectin was the earliest matrix protein expressed in calvariae, with a peak expression in fetal 16- and 17-day (d) bones. Fibronectin expression coincided with the condensation of preosteoblasts prior to calcification and decreased once bone mineralization commenced. The expression of type I collagen, osteonectin, bone sialoprotein, and alkaline phosphatase mRNAs was found at 17 d. The increase in type I collagen mRNA levels was correlated with a 3.5-fold increase in calcium deposition at 19-20 d. Bone sialoprotein and alkaline phosphatase peaked on fetal 21 d while osteonectin remained at a low level and was localized to the osteoblast layer and the osteocyte lacunae. Osteopontin mRNA levels increased rapidly in neonatal calvariae. After birth, osteonectin and fibronectin were mainly associated with blood vessels. Thus, fibronectin is one of the earliest matrix proteins expressed in calvariae and is rapidly followed by type I collagen, bone sialoprotein, and alkaline phosphatase. Osteocalcin, osteonectin, and osteopontin mRNAs have similar patterns of expression in the developing fetal calvaria, and their synthesis coincided with mineralization.