We investigated the effect of sustained release of bone morphogenetic protein-2 (BMP-2) from an injectable chitosan gel on osteoblastic differentiation in vitro. We first characterized the release profile of BMP-2 from the gels, and then examined the cellular responses of preosteoblast mouse stromal cells (W-20-17) and human embryonic palatal mesenchymal (HEPM) cells to BMP-2. The release profiles of different concentrations of BMP-2 exhibited sustained releases (41% for 2 ng/mL and 48% for 20 ng/mL, respectively) from the chitosan gels over a three-week period. Both cell types cultured in the chitosan gels were viable and significantly proliferated for 3 days (p < 0.05). Chitosan gels loaded with BMP-2 enhanced ALP activity of W-20-17 by 3.6-fold, and increased calcium mineral deposition of HEPM by 2.8-fold at 14 days of incubation, compared to control groups initially containing the same amount of BMP-2. In addition, schitosan gels loaded with BMP-2 exhibited significantly greater osteocalcin synthesis of W-20-17 at seven days, and of HEPM at both 7 and 14 days compared with the control groups (p<0.05). This study suggests that the enhanced effects of BMP-2 released from chitosan gels on cell differentiation and mineralization are species and cell type dependent. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2011.