To harness the potential of ES (embryonic stem) cells for human therapy, technology to develop the large-scale expansion and differentiation of these cells is required. In the present study, we tested various conditions for the expansion and neural commitment of mouse ES cells, using a cell line with a fluorescent reporter, which allows the monitoring of these processes by flow cytometry. The expansion of the 46C ES cell line in the presence of two different media [serum-free ESGRO Completetrade mark and DMEM (Dulbecco's modified Eagle's medium) containing 10% (v/v) fetal bovine serum] was compared. Both media yielded similar cell fold increases at two different initial cell densities and were able to maintain neural commitment potential during expansion. The influence of inocula concentration in the presence of two different media on cell proliferation and efficiency of neural commitment was evaluated. Two different chemically defined serum-free media were tested: the more conventional N2B27 and the second-generation medium RHB-A (Stem Cell Sciences, Edinburgh, Scotland, U.K.). The kinetics of neural commitment was followed during 8 days in the presence of both media. Our results show that inocula concentrations between 5x10(3) and 10(4) cells/cm(2) are the most appropriate to achieve a better cell growth and more efficient neural commitment. We also show that cell culture in RHB-A medium results in higher rates of cell proliferation and neural commitment of ES cells, when compared with N2B27.