Memory B cells (MBCs) are long-lived sources of rapid, isotype-switched secondary antibody-forming cell (AFC) responses. Whether MBCs homogeneously retain the ability to self-renew and terminally differentiate or if these functions are compartmentalized into MBC subsets has remained unclear. It has been suggested that antibody isotype controls MBC differentiation upon restimulation. Here we demonstrate that subcategorizing MBCs on the basis of their expression of CD80 and PD-L2, independently of isotype, identified MBC subsets with distinct functions upon rechallenge. CD80 + PD-L2 + MBCs differentiated rapidly into AFCs but did not generate germinal centers (GCs); conversely, CD80-PD-L2- MBCs generated few early AFCs but robustly seeded GCs. The gene-expression patterns of the subsets supported both the identity and function of these distinct MBC types. Hence, the differentiation and regeneration of MBCs are compartmentalized.