In the normal immune system, B cells are thought to be negatively or positively selected at various checkpoints during their maturation; a process that maintains a broad immunoglobulin repertoire while eliminating non-functional or potentially harmful autoreactive antibodies. This study tested the hypothesis that utilization of certain immunoglobulin heavy chain variable region (VH) genes, possibly as a consequence of intrinsic affinity for various ligands, directs positive or negative B cell selection coupled to B cell activation in the periphery during the immune response. The specific prediction that the VH repertoire of CD40-activated B cells would differ from the repertoire of unstimulated cells from the same donor, was tested by assessing VH utilization among human B cell clones grown in vitro, following stimulation with CD40 ligand (CD40L) and IL-4. The results showed that, although utilization of the known VH families and of individual VH3 genes was similar to that found in unstimulated B lymphocytes of the same donor, utilization of individual VH4 genes in CD40-activated B cells displayed a pattern that was markedly different from that of the unstimulated B cells. An allele of V4-61, V4-61b, was over-represented among the activated cells and, in contrast, the V4-34 gene (known to encode cold agglutinins with strong autoreactive properties) was modestly represented among the VH4 activated B cells, although V4-34 was overwhelmingly predominant in the repertoire of resting B cells. These results point to the existence of selection mechanisms that operate during B cell activation in the periphery. These mechanisms may favor B cells utilizing certain VH genes and disfavor the cells that utilize other genes, possibly because utilization of the latter confers autoreactivity.