Nuclear factor-kappaB (NF-kappaB) plays a crucial role in B-cell and lymphoid organ development. Here, we studied the consequences of constitutive, signal-independent activation of the alternative NF-kappaB pathway for the splenic marginal zone (MZ). In contrast to nfkb2(-/-) mice, which lack both p100 and p52, mice that lack only the inhibitory p100 precursor but still express the p52 subunit of NF-kappaB2 (p100(-/-)) had markedly elevated MZ B-cell numbers. Both cell-intrinsic mechanisms and increased stromal expression of vascular cell adhesion molecule-1 (VCAM-1) contributed to the accumulation of MZ B cells in p100(-/-) spleens. While migration of p100(-/-) MZ B cells toward the lysophospholipid sphingosine-1 phosphate (S1P) was not affected, CXCL13-stimulated chemotaxis was impaired, correlating with reduced migration of MZ B cells into follicles in response to lipopolysaccharide (LPS). Strikingly, p100 deficiency resulted in the absence of a normal marginal sinus, strongly induced expression of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and glycosylated cell adhesion molecule-1 (GlyCAM-1), and the formation of nonfunctional ectopic high endothelial venule (HEV)-like structures in the red pulp. Thus, constitutive activation of the alternative NF-kappaB pathway favors MZ B-cell development and accumulation but leads to a disorganized spleen microarchitecture.