The effects of creatine (Cr) supplementation on primary rat osteoblast-like cells cultured as monolayer and micromass were investigated. Cr was added to the medium at concentrations of either 10 or 20 mM. At various time points, the cell cultures were analyzed morphologically, metabolically and biochemically. The degree of differentiation of primary osteoblast-like cell cultures was higher in micromass cultures compared to monolayer cultures, as judged by alkaline phosphatase (ALP) activity and extent of mineralization. In both culture systems, Cr supplementation showed positive effects, which were dependent on the organizational level of the osteoblast-like cells in such a way that the cells in monolayer culture showed significantly increased metabolic activity, ALP activity and mineralization in the presence of Cr than without the supplement. In micromass cultures, Cr also significantly enhanced ALP activity and mineralization, without affecting metabolic activity. The effect of Cr on ALP activity was more pronounced at higher concentrations of Cr, but 20 mM Cr already showed some adverse effects on cell viability. In conclusion, chemically pure Cr added to low serum cell culture medium has a stimulatory effect on metabolic activity, differentiation and mineralization of osteoblast-like cells indicating that Cr supplementation could also be used as a potential clinical intervention to stimulate cell growth, differentiation and mineralization during bone repair in vivo.