This study examined the osteoblast-like MC3T3-E1 cell responses to poly(DL-lactide) (PDLLA) and poly(L-lactide) (PLLA) with different weight average molecular weight (M.W.). Colony formation of MC3T3-E1 cells on the PLLA with M.W. 270000 or 1370000 was slightly lower than that on glass. The protein, DNA and hydroxyproline (HYP) content and alkaline phosphatase (ALP) activity for cells cultured on the PLLA (M.W. 270000 or 1370000) for 14 d were almost similar to those on glass. In contrast, the ALP activity of the cells cultured on low M.W. PLLA (M.W. 20000) increased. Osteoblast differentiation was stimulated by low M.W. PLLA but not by high M.W. PLLA. The addition of low M.W. PDLLA (M.W. 5000 or 10000), L-lactide or L-lactic acid into culture increased the protein, DNA and HYP content and ALP activity for cells at 100 microg/ml. Compared with four chemicals, PDLLA (M.W. 10000) had the strongest simulation effect on the cell. The release of L-lactic acid from PLLA and PDLLA into aqueous solution during incubation only slightly affected cell activity. In a cell-free condition, in the presence of PDLLA, the ALP activity was maintained without inactivation, even after 24 h incubation. Such a phenomenon was not seen with L-lactide and L-lactic acid. This may be a reason why PDLLA has a stronger effect on osteoblast differentiation relative to L-lactic acid. These results suggested that increased osteoblast differentiation was induced by low M.W. PDLLA and PLLA, and these may be used as a effective material in the field of orthopedic and drug delivery systems for the treatment of bone diseases.