In general, cell response is affected by both chemical composition and surface roughness of implant materials. The aim of this study was to evaluate the effect of titanium (Ti) chemical composition and surface roughness on the response of rat bone marrow cells, examining cell attachment, cell proliferation, total protein content, alkaline phosphatase (ALP) activity, and bone-like nodule formation. Cells were cultured on both commercially pure titanium (cpTi) and titanium-6-aluminum-4-vanadium alloy (Ti-A) discs with four different average roughnesses (Ra). For attachment evaluation, cells were cultured for 2 h. After 14 days, cell proliferation, total protein content, and ALP activity were evaluated. Bone-like nodule formation was evaluated after 21 days. Data were compared by anova and Duncan's multiple range test when appropriate. Cell attachment and total protein content were affected by neither Ti chemical composition (P = 0.201, and P = 0.639, respectively) or surface roughness (P = 0.972, and P = 0.660, respectively). Proliferation, ALP activity, and bone-like nodule formation were affected only by Ti chemical composition (P = 0.0001, P = 0.064, and P = 0.0001, respectively). These results suggest that cpTi would optimize osteoblastic differentiation by rat bone marrow cells, including reduced cell proliferation, and increased ALP activity and bone-like nodule formation, while surface roughness, within the Ra parameters used, would not affect significantly the rat bone marrow cell response.