Circulating leukocytes are thought to extravasate from venules through open interendothelial junctions. To test this paradigm, we injected N-formyl-methionyl-leucyl-phenylalanine (FMLP) intradermally in guinea pigs, harvesting tissue at 5-60 min. At FMLP-injected sites, venular endothelium developed increased surface wrinkling and variation in thickness. Marginating neutrophils formed contacts with endothelial cells and with other neutrophils, sometimes forming chains of linked leukocytes. Adherent neutrophils projected cytoplasmic processes into the underlying endothelium, especially at points of endothelial thinning. To determine the pathway by which neutrophils transmigrated endothelium, we prepared 27 sets of serial electron microscopic sections. Eleven of these encompassed in their entirety openings through which individual neutrophils traversed venular endothelium; in 10 of the 11 sets, neutrophils followed an entirely transendothelial cell course unrelated to interendothelial junctions, findings that were confirmed by computer-assisted three-dimensional reconstructions. Having crossed endothelium, neutrophils often paused before crossing the basal lamina and underlying pericytes that they also commonly traversed by a transcellular pathway. Thus, in response to FMLP, neutrophils emigrated from cutaneous venules by a transcellular route through both endothelial cells and pericytes. It remains to be determined whether these results can be extended to other inflammatory cells or stimuli or to other vascular beds.