We describe an automated, observer-independent and highly reproducible assay for the quantification of transmigrated neutrophils across endothelial monolayers. Endothelial cells grown on collagen gels were loaded with a dye emitting red fluorescence. Neutrophils loaded with dye emitting green fluorescence were allowed to adhere to and transmigrate across endothelial monolayers. For quantification of adherent and migrated cells, randomly selected fields were scanned by confocal laser scan microscopy at defined depths within and below the endothelial monolayers. The images obtained were transferred into the public domain NIH image program and numbers and distribution of cells within scanned sectors were automatically calculated. We demonstrate that adherent neutrophils are easily discriminated from transmigrated cells; absolute numbers of migrated cells can be reproducibly calculated by counting cells at a depth of -20 microm, thus permitting evaluation of large-scale experiments: the efficacy of neutrophil transmigration depends on the level of endothelial activation after TNF stimulation and mAbs to cell surface adhesion molecules interfere with migration in a manner similar to that previously shown in in vivo experiments. This assay lends itself to the identification of molecules influencing in cell migration in each phase of EC activation and to the screening of pro- and anti-migratory properties of biological or pharmacological reagents.