Nanofibrous scaffolds were prepared from gelatin solutions and were further cross-linked with glutaraldehyde (GA). The fiber diameter was varied from 100 to 1000 nm by controlling the applied voltage (4-15 kV) and the concentration of the gelatin solution (4-15%). The tensile moduli and the tensile strength of the noncross-linked scaffolds varied from 20 to 120 MPa and 0.5 to 3.5 MPa, respectively. Crosslinking with GA led to an increase in both the tensile modulus and strength and correlated with cross-linker concentration. Gelatin-based matrices were characterized by Fourier transform infrared spectroscopy and differential scanning calorimetry. High cellular viabilities and rounded morphology of chondrocytes was observed at the end of 7 days in culture with added matrix deposition and flattening of cells at 15 days. Matrix stiffness was noted to impact cell densities and the expression of chondrocytic markers, especially aggrecan. The ratios of collagen-II (C-II) to collagen-I (C-I) of 0.62 and 1.33 were noted on gelatin nanofibrous scaffolds cross-linked with 0.1% GA at the end of 7 and 15 days in culture, respectively. C-II/C-I ratios of 1.30 and 2.58 were noted on scaffolds cross-linked with 1.0% GA at the end of 7 and 15 days in culture, respectively. (C) 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 95A: 828-836, 2010.