The objective of the study was to analyze and compare the abilities of various human cell types with inherently dissimilar osteogenic potentials to induce heterotopic bone formation following ex vivo transduction with two distinct adenoviral vectors encoding bone morphogenetic protein type 2 (BMP2). The cells comprised primary human bone marrow mesenchymal stem cells (BM-MSCs), primary human skin fibroblasts (SFs), and a human diploid fetal lung cell line (MRC-5). The vectors included adenovirus type 5 or a chimeric adenovirus type 5 with the fiber gene of adenovirus type 35 (Ad5F35-BMP2), both demonstrating significantly different expression of BMP2 in vitro. The experimental groups consisted of the three human cell types transduced with each of the two adenoviral vectors. Using nonobese diabetic severe combined immunodeficiency (NOD/SCID) mice, the transduced cells were injected intramuscularly following ex vivo adenoviral transduction. The nature and extent of heterotopic bone formation were analyzed radiographically and histologically. At 14 days postinjection, abundant, highly mineralized bone was formed in mice injected with Ad5F35-BMP2-transduced cells irrespective of the cell type. There was no statistically significant difference in the amount of bone formed between BM-MSCs, SFs, and MRC-5 cells transduced with Ad5F35-BMP2, as assessed from bone surface area on biplanar plain radiography. Substantially lesser amounts or no bone could be detected in mice injected with cells transduced with Ad5-BMP2. Immunohistochemical analysis confirmed the presence of human cells in muscle as early as 2 days postdelivery; however, at 6-7 days after injection, the transduced cells could not be detected in surrounding muscle, or in the heterotopic bone, indicating the host origin of the newly formed bone. The results of the study demonstrate no significant difference in osteoinductive properties between BM-MSCs, SFs, and MRC-5 cells transduced ex vivo with the same type of adenovirus encoding BMP2. The level of BMP2 expression appears to be a crucial factor determining the extent of heterotopic bone formation and was significantly affected by the type of adenovirus used. In the cell types studied, Ad5F35-BMP2 was more efficacious than Ad5-BMP2 in providing adequate levels of BMP2 for efficient osteoinduction.