The splenic marginal zone (MGZ), which surrounds the mantle zone (MTZ) in human splenic white pulp, contains a phenotypically and morphologically distinct population of B cells. The origin of MGZ B cells is uncertain. Whereas some experiments in rodents have suggested that they are a distinct cell lineage responsible for the immune response to T-independent type 2 antigens, others have suggested that they are memory B cells derived from a germinal center (GC) response. The progeny of a GC reaction is expected to have rearranged immunoglobulin (Ig) genes that are mutated. The distribution of mutations would be expected to reflect the selection of Ig by its affinity for antigen. We have analyzed rearranged Ig heavy chain variable region (VH) 6 and VH 4.21 genes in MGZ and MTZ B cells microdissected from frozen sections of human spleen to determine whether these genes have the properties of an affinity-selected memory B cell population. MTZ B cells contained germline Ig VH genes, confirming previous reports and providing an internal control for mutational analysis. MGZ B cells contained Ig VH genes that were mutated, showing that these cells had been subjected to a mutational mechanism characteristically active in the GC. The rearranged VH 6 genes showed patterns of mutation indicative of an antigen selection process, whereas the distribution of mutations in VH 4.21 genes was not characteristic of gene selection by conventional T-dependent antigen. Our studies provide the first evidence that the human splenic MGZ is a reservoir of memory B cells.