While a number of chemoattractants of vascular endothelial cells have now been identified in vitro, differences in methodology preclude comparisons of substances evaluated in different assays. Here, we report a standardized chemotactic assay in which the migration of calf pulmonary artery endothelial cells in a 48-well microchemotaxis chamber was determined. Nonstimulated (control) migration was remarkably constant (mean +/- SD, 96 +/- 14) from plate to plate, thus allowing the indexing of relative migration of stimulated cells to that of nonstimulated cells in the control wells of that plate. Based on the relative migrations observed in response to each of the substances evaluated, those proving to be stimulatory of migration were placed in rank order by potency. The growth factors epidermal growth factor, transforming growth factor-alpha, and basic fibroblast growth factor (followed by pentosan polysulfate, plasmin, fibronectin, fibrinogen, granulocyte-macrophage colony stimulating factor heparin, adenosine, and MgSO4) were the most potent. Only the platelet factors platelet-derived growth factor-BB and platelet activating factor proved inhibitory of migration. Combining fibrinogen with other chemoattractants produced either stimulation or inhibition in comparison to the migration observed with fibrinogen alone, suggesting that more than one signal transduction mechanism was, in all likelihood, invoked by the various agents. This assay will allow the rapid screening and rank ordering of additional putative chemoattractants, will facilitate the study of the biochemical mechanisms involved in endothelial cell migration, and will permit the evaluation of pharmacologic agents capable of modulating stimulated or unstimulated migration.