In this in vitro study, we compared the cytocompatibility and osteoblast promoting potency on human bone marrow cell culture with three different alloys (surgical steel, CoCr, Ti6Al4V) and three different surface structures (polished, sandblasted, porous coated). These biometals were specifically chosen because of their current applications in clinical orthopedic practices. Human mononuclear bone marrow cells were cultivated onto the surface of the different biomaterials and stimulated by dexamethasone, L-ascorbic-acid-2-phoshpate and beta-glycerolphosphate over a 3-week period. Immunofluorescent stainings against several antigens (ALP, RANKL, osteopontin, collagen I), mRNA-expression of collagen (Col) I/II, BSP, osteopontin, osteocalcin, TRAP, light and scanning electron microscopy evaluation were used to evaluate cellular growth and osteoblast differentiation. For surface roughness and energy analysis of the specimen, roughness profile (Ra, Rz) and contact angle (CA) measurements were performed. We found differences between the different biometals and surface structures. Steel showed potential cytotoxic effects whereas CoCr and more Ti6Al4V showed an excellent cytocompatibility. There were no qualitative differences in mRNA expression between each of the tested biomaterials. In terms of antigen expression, a sandblasted Ti6Al4V surface showed enhanced osteoblastic differentiation. A porous-coated surface improved the osteoconductivity of CoCr when compared to a polished surface. In contrast to controls all cells cultivated with biometals induced a RANKL expression. Cells increased the implant roughness with the exception of sandblasted Ti6Al4V. Our data show that surface topography and physicochemical properties of biometals influence osteoblast differentiation in vitro.