The macrophage scavenger receptor macrophage receptor with a collagenous structure (MARCO) is expressed in mice by the marginal zone macrophages of the spleen and by macrophages of the medullary cords of lymph nodes, as well as the peritoneal macrophages. MARCO is a relative of scavenger receptor A (SR-A), the more widely expressed prototypic member of the scavenger receptor family. In the present study, we found that genetic ablation of MARCO leads to changes in the organization of the splenic marginal zone, and causes a significant reduction in the size of the resident peritoneal macrophage population, possibly due to changes in adhesion and migration capacity. In mice lacking both MARCO and SR-A these effects are even more apparent. During ontogeny, the appearance and organization of the MARCO-expressing cells in the spleen precedes the appearance of other receptors on macrophages in the marginal zone, such as SIGNR1 and Siglec-1. In the absence of MARCO, a clear delay in the organization of the marginal zone was observed. Similar findings were seen when the reappearance of the various subsets from precursors was studied after depleting macrophages from the adult spleen by a liposome treatment. When challenged with a pneumococcal polysaccharide vaccine, a T-independent type 2 Ag for which an intact marginal zone is crucial, the knockout mice exhibited a clearly impaired response. These findings suggest that both MARCO and SR-A, in addition to being important scavenger receptors, could be involved in the positioning and differentiation of macrophages, possibly through interaction with endogenous ligands.