The alteration in bone components in the femoral-diaphyseal tissues with fracture healing was investigated. Rats were sacrificed 7 and 14 days after the femoral fracture. Protein content in the femoral-diaphyseal tissues was markedly elevated by fracture healing. Analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that many protein molecules were induced in the diaphyseal tissues with fracture healing. Moreover, when the femoral-diaphyseal tissues with fracture healing were cultured for 24 and 48 h in a serum-free medium, many proteins in the bone tissues were released into the medium. Also, the culture of the diaphyseal tissues with fracture healing caused a significant increase in bone alkaline phosphatase activity and deoxyribonucleic acid (DNA) content. Meanwhile, the presence of zinc acexamate (10-5 and 10-4 M), a stimulator of bone formation, in a culture medium induced a significant elevation of protein content and alkaline phosphatase activity in the diaphyseal tissues with fracture healing. Such an effect was completely abolished by the presence of cycloheximide (10-6 M), an inhibitor of protein synthesis. The present study suggests that fracture healing induces a newly synthesized bone protein component including stimulatory factor(s) for bone formation. Zinc supplementation may stimulate the healing of femoral fracture.