The present study was performed to investigate morphologically the process of neutrophil extravasation induced by the synthetic bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) in venules of the rat mesentery by the combined use of intravital microscopy and transmission electron microscopy (TEM). Special attention was given to the interaction of the neutrophils with the endothelial cells and endothelial basal lamina. By intravital microscopy, the rolling and adhesion of leukocytes were observed within 3 min in preparations activated by fMLP. Neutrophils remained in the vascular wall for more than 30 min even after penetration of the endothelium. In this period, neutrophils migrating between endothelial cells and their basal lamina were often observed by TEM. After 40 min application of fMLP, some of the migrating neutrophils parted from the vessel wall into the surrounding connective tissues. There were neutrophils which passed through the small pore of the basal lamina with a cytoplasmic constriction. These findings indicate that the endothelial basal lamina acts as a physical barrier against neutrophil extravasation, thus resulting in the transient retainment of neutrophils beneath the endothelial cells of the venular wall.