Lymphocyte trafficking into Peyer's patches requires beta 7 integrins and L-selectin. Here, we use intravital microscopy to examine leukocyte rolling and adhesion in Peyer's patch high endothelial venules (HEV) of wild-type, L-selectin-deficient (L-/-), beta 7 integrin-deficient (beta 7-/-), and beta 7/L(-/-) mice. Although the leukocyte rolling flux fraction was reduced by 70%, Peyer's patches in L-/- mice were of normal size and cellularity. In beta 7-/- mice, the rolling flux fraction was normal, but the number of adherent leukocytes in HEV was greatly reduced. The median leukocyte rolling velocity was reduced in L-/- mice and increased in beta 7-/- mice, suggesting that beta 7 integrins and L-selectin mediate rolling in Peyer's patch HEV at different velocities. beta 7/L(-/-) exhibited both a low rolling flux fraction and low adhesion and had severely reduced Peyer's patch size and cellularity. The residual rolling in these mice was completely blocked by a P-selectin mAb. A significant P-selectin component was also detected in the other genotypes. Twenty-six percent of B and T lymphocytes isolated from Peyer's patches of wild-type mice expressed functional ligands for P-selectin, and this fraction was increased to 57% in beta 7/L(-/-) mice. Peyer's patch HEV were found to express P-selectin under the conditions of intravital microscopy, but not in situ. Our data suggest a novel P-selectin dependent mechanism of lymphocyte homing to Peyer's patches. In situ, beta 7 integrins and L-selectin account for all lymphocyte homing to Peyer's patches, but P-selectin-dependent rolling, as induced by minimal trauma, may support trafficking of effector T lymphocytes to Peyer's patches.