The effect of zinc, a stimulator of bone formation, on bone protein components in the femoral-diaphyseal tissues with fracture healing was investigated. Rats were sacrificed between 1 and 7 days after the femoral fracture, and the diaphyseal tissues were cultured in a serum-free Dulbecco's modified Eagle's medium for 24 h. Protein content in the femoral-diaphyseal tissues was markedly elevated by fracture healing. The amount of protein in the medium cultured with the diaphyseal tissues obtained from fracture healing rats was markedly elevated as compared with that of normal rats, indicating that bone protein components were secreted into culture medium. Analysis with sodium dodecyl sulfate-polyacrylamide gel elecrophoresis (SDS-PAGE) showed that many protein molecules were secreted from the diaphyseal tissues with fracture healing. Especially, protein molecule of about 66 kDa was markedly secreted by fracture healing. The presence of zinc acexamate (10(-5) and 10(-4) M) in culture medium induced a significant elevation of medium protein content; the zinc effect was enhanced by culture with the diaphyseal tissues of fracture healing rats. Also, the culture of diaphyseal tissues with fracture healing caused a significant increase in insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-beta1) in culture medium. The production of IGF-I and TGF-beta1 from bone tissues with fracture healing was significantly enhanced in the presence of zinc acexamate (10(-6)-10(-4) M). Moreover, the addition of IGF-I (10(-8) M) or TGF-beta1 (10(-10) M) in a culture medium caused a significant elevation of protein content in the medium cultured with the femoral-diaphyseal tissues from normal and fracture healing rats. The effect of IGF-I or TGF-beta1 was significantly enhanced in the presence of zinc acexamate (10(-4) M). Also, deoxyribonucleic acid (DNA) content in the diaphyseal tissues from normal and fracture healing rats was significantly raised by the culture with IGF-I or TGF-beta1. The effect of IGF-I was significantly enhanced by zinc. The present study demonstrates that fracture healing increases production of bone IGF-I and TGF-beta1, and that this elevation is enhanced by zinc treatment.