Interactions between peripheral blood mononuclear cells (PBMC) and the endothelium critically determine vascular repair and reparative angiogenesis, but also pathological processes, such as atherosclerosis. Current methodology to study these interactions mostly regards PBMC as a homogenous population or it restricts its focus on individual cell types and therefore does not appreciate the differential behavior of individual PBMC subpopulations, which synergize or antagonize each other to obtain the overall effect. We therefore developed a flow cytometry-based in vitro assay to assess multiple parameters of interaction between several individual populations of PBMC and an endothelial monolayer. Freshly isolated, unlabelled human PBMC were left to adhere to or transmigrate through a monolayer of fluorescence-labeled human aortic endothelial cells grown to confluence on the filter membrane of sterile transwell migration inserts. Monocyte chemoattractant protein-1 (MCP-1) was applied as a chemoattractant to the lower compartment of the migration chamber. After 6 h, transmigrating PBMC were harvested from the lower compartment, while nonadherent and adhering cell populations were harvested from the upper compartment by sequential washing/detachment. All three cell fractions were then individually stained with fluorescence-labeled monoclonal antibodies and analyzed by flow cytometry. Quantification was achieved by the usage of counting beads. Endothelial cells were separated from PBMC during the analysis by a multiparametric gating strategy. Using the newly established assay, we observed distinct migration patterns for inflammatory CD14(hi) CD16(neg) and resident CD16(pos) monocytes. These cell types differed in their basal adhesion and transmigration patterns as well as their responses to the CCR2 ligand MCP-1. This assay allows for the parallel study of interactions between multiple individual leukocyte populations and an endothelial layer. Several readouts can be derived from the same experiment, like the composition of adhering and transmigrating cell fractions or the individual adhesion/migration behavior of several distinct cell types.