Because isolation of sufficient numbers of cycling, germinal center B cells from mice for biochemical characterization of BCR-derived signals can be problematic, we have designed an experimental approach for generating large numbers of cycling B cells for further study. In the experiments reported here, small, resting B cells were polyclonally stimulated with lipopolysaccharide (LPS), and cycling B cells isolated as two bands on three-step Percoll gradients. Cycling B cells isolated at Days 2, 4, or 6 of preactivation showed an increased expression of Fas receptor and peanut agglutinin binding, with a concomitant decrease in sIgD positivity. These cells phenotypically resembled extrafollicular or early germinal center B cells. These cycling B cells were used to study the functional consequences of differential signaling through the BCR. Strong cross-linking of BCR, by restimulation of cycling normal B cells with either immobilized or soluble F(ab')2 anti-mu and cycling hen egg lysozyme (HEL) transgenic B cells with either soluble or immobilized HEL, extended cellular proliferation by 2-3 d. In contrast, cycling B cells either restimulated with soluble, whole anti-mu (to mimic binding of soluble immune complexes) or cultured in the absence of restimulation (to mimic cycling B cells not competitive for antigen) resulted in the rapid exit of the cells from cycle. This system will enable the molecular and biochemical characterization of signal delivery to cycling B cells.