Bone formation was studied after intramuscular implantation of demineralized bone matrix. Ash weight determinations were used to verify the bone-forming ability of implants, and confirmed that no bone was formed when nonactive implants (stripped of their bone-forming ability) were used. A solution hybridization/RNase protection assay was used for the detection of specific mRNA transcripts in the implants and surrounding tissue. Analysis of insulin-like growth factor I (IGF-I) mRNA showed a transient increase peaking on day 3 following implantation. Radioimmunoassay (RIA) for IGF-I-like immunoreactivity indicated a corresponding increase of IGF-I peptide in extracts from the implants at that time point. IGF-II mRNA and alkaline phosphatase mRNA reached highest levels around day 11 following implantation. Bone formation in old rats, 50 weeks of age, was associated with lower IGF-I mRNA levels 3 days after implantation compared with young animals. IGF-II mRNA levels were also affected and tended to be higher 12 days after implantation compared with young animals. These results indicate that IGFs could be paracrine or autocrine factors in the bone-forming process. During this process, IGF-I mRNA is expressed at an early stage, in correlation with the recruitment and proliferation of surrounding mesenchymal cells, whereas IGF-II mRNA is activated significantly later, correlating to the beginning of the actual calcifying process during endochondral bone formation.