Endothelial cells play a crucial role in maintaining cardiovascular homeostasis. Although many cardiovascular disorders involve endothelial cell dysfunction, the specific cellular and molecular mechanisms involved are not well known. We sought to establish a reproducible method of endothelial cell isolation from gene targeted mice to specifically examine endothelial pathophysiological mechanisms. Primary aortic endothelial cell cultures were established from wild type and intercellular adhesion molecule-1 (ICAM-1) deficient mice. Isolation of mouse aortic endothelial cells (MAEC) by fluorescent activated cell sorting routinely resulted in pure, homogenous, primary cultures. Wild type and ICAM-1 deficient endothelial cell morphology was similar, with both cultures showing cobblestone morphology and DiI-Ac-LDL staining. Monocyte adhesion to ICAM-1 deficient aortic endothelial cells was decreased by 86% as compared with wild type MAEC. Monocyte adhesion was also determined using YN-1, an ICAM-1 blocking antibody. YN-1 decreased monocyte adhesion to wild type aortic endothelial cells by 25%, whereas YN-1 did not further decrease monocyte adhesion to ICAM-1 deficient MAEC. These data demonstrate that gene targeted endothelial cell cultures are an effective means of identifying specific cellular and molecular mechanisms involved in endothelial cell physiology and dysfunction.