Polyclonal B-cell activation is the central theme in the production of autoantibodies and possible activation of autoreactive T cells in both human and murine lupus. The abnormal expansion of CD5 þ B cells in murine lupus has been suggested, in particular, to be one of the most characteristic findings in these mice. Activated B cells can be separated from the B cells of resting stage by the difference in cell density. The aim of this study was to investigate the characteristics of different densities of the spleen cells separated by gradient density. Furthermore, the ability of anti-DNA antibody secretion in each percoll gradient fraction of B cells was also analysed. The results showed: a higher percentage of CD5 þ B cells, which corresponded to the activated Bcell population, in percoll gradient 1 and 2 fractions; that splenic B cells of NZB/W F 1 mice had proliferative response to interleukin (IL)-4 or IL-5 but not to IL-10 or interferon-g (IFN-g); and that B cells isolated by percoll gradient produced anti-DNA antibody after stimulation with lipopolysaccharide (LPS) plus IL-5 and IFN-g, but not IL-4 and IL-10. These data suggest that B cells at different stages of activation express differential characteristics and functions.