Transforming growth factors type β (TGF-β1, -β2, and -β3) are potent stimulators of bone formation and have been shown to regulate chondrocyte, osteoblast, and osteoclast formation and function. However, the distribution of the different isoforms and their signaling receptors in human bone in vivo has not previously been reported. Using samples of normal (neonatal rib) and pathological (osteophytic) developing human bone, we have investigated the expression of the different TGF-β isoforms and their signaling receptors (TGF-βRI and RII) at the messenger ribonucleic acid (mRNA) and protein levels by in situ hybridization and immunolocalization to establish the sites of TGF-β production and their possible sites of action during human bone development in vivo. All three TGF-β isoforms and the receptors were detected at sites of endochondral and intramembranous ossification. At sites of endochondral ossification, TGF-β2 was detected in all zones of the cartilage, with the highest expression seen in the hypertrophic and mineralizing zones. TGF-β3 was detected in proliferative and hypertrophic zone chondrocytes, while TGF-β1 expression was restricted to the proliferative and upper hypertrophic zones. TGF-βRI and RII exhibited similar distributions with maximum expression in the hypertrophic and mineralizing zones in the neonatal rib but in the resting/proliferative zone in the developing osteophyte. At sites of intramembranous ossification TGF-β3 was the most widely distributed isoform and showed both matrix- and cell-associated staining. TGF-β2 and -β1 were expressed almost exclusively at sites of mineralization. These observations demonstrate that the different TGF-β isoforms and their receptors exhibit distinct but overlapping patterns of expression, and support the hypothesis that they are involved in the regulation of endochondral and intramembranous ossification during human bone development in vivo.