The interaction of antigen with surface Ig receptors initiates a complex process by which resting B lymphocytes are activated. In this study, small resting B lymphocytes were isolated by countercurrent elutriation of tonsillar B lymphocytes and stimulated with a range of concentrations of anti-mu and B cell growth factor (BCGF). The subsequent expression of two glycoproteins known to be absent from resting B lymphocytes but present on activated cells were analyzed with the monoclonal antibodies 4F2 and 5E9. The antigen recognized by the 4F2 monoclonal was expressed early in the G1 phase of the cell cycle and correlated with blast transformation; this was demonstrated by stimulating resting B cells with a low concentration of anti-mu sufficient for cellular activation but not proliferation. The 5E9-defined antigen was not present unless the B cells were stimulated with a higher concentration of anti-mu or a BCGF was added to the low concentration of anti-mu. The addition of hydroxyurea to culture blocked the entrance of stimulated B cells into S phase but did not interfere with the expression of either 4F2 or 5E9. Thus, whereas the monoclonal antibody 4F2 recognized cells in early G1, 5E9 recognized cells in late G1. By double immunofluorescence staining with propidium iodide and either 4F2 or 5E9, all cells in S, G2, and M phases were demonstrated to be both 4F2- and 5E9-positive. The monoclonal antibodies 4F2 and 5E9 can be used to distinguish an activated from a resting human B lymphocyte and to delineate sequential steps in the activation process.