Grafting negatively charged groups such as phosphates is a well-known strategy for inducing the deposition of apatite-like layers under simulated physiological conditions. In this investigation, chitosan was phosphorylated in an attempt to enhance its osteoconduction. Chitosan membranes were phosphorylated at 30 degrees C with the H3PO4/Et3PO4/P2O5/butanol reaction system for periods up to 48 h. This method is an alternative to the phosphoric acid/urea/dimethylformamide phosphorylation method, which involves the use of much higher temperatures. In this study, the effects of the phosphorylation reaction time on the surface morphology and surface free energy of phosphorylated membranes were investigated with atomic force microscopy and static-contact-angle measurements, respectively. In addition, the modified membranes were evaluated with respect to their cytotoxicity toward bone cells through the incubation of human osteoblastic cells with extracts of the materials for two different periods: 24 and 120 h. The results revealed a reduction of the average surface roughness at the nanometer scale with increasing phosphorylation reaction time. Wettability studies showed an increase in the polar component of the surface free energy with increasing reaction time as a result of the increase in the phosphate surface concentration. Cytotoxicity studies revealed no cytotoxic effect of phosphorylated membranes on osteoblastic cells, regardless of the incubation period. (c) 2006 Wiley Periodicals, Inc.