Cultured rabbit aortic and human carotid artery endothelial cells produced a factor that was chemotactic for monocytes but not for neutrophils. Checkerboard analysis showed that the activity was due to chemotaxis and not to chemokinesis. The factor was produced in both serum-containing and serumless media. Treatment with carboxypeptidase and trypsin resulted in inhibition of chemotactic activity, indicating that the factor is a peptide. Medium from cultures of rabbit aortic, human carotid artery, and human umbilical vein endothelial cells previously exposed to beta migrating very low density lipoprotein (beta-VLDL) had substantially more chemotactic activity than medium from untreated cells or cells exposed to low density lipoprotein. beta-VLDL alone had no chemotactic activity. We conclude that large vessel endothelial cells produce a monocyte chemotactic factor that is increased after exposure of the cells to beta-VLDL.