Recombinant human growth/differentiation factor 5 (rhGDF-5) and recombinant human bone morphogenetic protein 2 (rhBMP-2), as members of the transforming growth factor ß family, influence bone formation and differentiation. This in vitro osteoblast cell culture study investigated the molecular biologic effect of these growth factors on regulator gene expression of the homebox proteins MSX1 and MSX2 as well as distal-less homebox 5 (Dlx5) and runt-related transcription factor 2 (Runx2/Cbfa1). Concerning effector genes, the messenger ribonucleic acids of osteocalcin (OCN) were quantified using a reverse transcriptase realtime polymerase chain reaction. Over a period of 15 days, osteoblasts were stimulated and analyzed at days 1, 2, 5, 10, and 15. rhGDF-5 and rhBMP-2 were applied in concentrations of 100, 500, and 1,000 ng/mL. The results showed enhanced gene expression of MSX1 and MSX2 by the lower rhGDF-5 concentration (100 ng/mL) during the first 48 hours and a marginally enhanced gene expression of Runx2 and OCN in a dose-dependent manner. The rhBMP-2 stimulation showed enhanced MSX1 and MSX2 gene expression with peaks at 24 and 240 hours; Runx2 and OCN were more highly expressed than the unstimulated control with the 100-ng/mL concentration. rhGDF-5 seems to stimulate early osteoblast differentiation and extracellular matrix production, while rhBMP-2 seems to boost early and late osteoblast differentiation. Low growth factor concentrations appeared to be more effective in terms of gene expression.