The effects of exogenous leukotrienes B4 and E4 (LTB4, LTD4) on the under-agarose motility of isolated normodense human eosinophils and neutrophils were examined using a novel sampling strategy for quantitation of leukocyte migration distance and vectorial orientation. Eosinophil chemotaxis to LTD4 was evident at a 10(-10)M threshold. The selective peptide-LT antagonist, SK&F 104353, abolished LTD4-induced eosinophil migration, indicating pharmacological specificity of the response. Neutrophil chemotaxis was apparent only with a very high (10(-5)M LTD4 concentration. LTB4 was a potent eosinophil and neutrophil chemoattractant over a 10(-9)M to 10(-4)M dose range. Analysis of leukocyte orientations provided evidence that chemokinetic responses were not being interpreted as indications of chemotactic behavior. LTB4 and LTD4 significantly altered neutrophil vectorial orientation. Comparison of migration distance and orientation at the leading edge and at the periphery of the well seeded with cells suggested that cell polarization appeared to be the earliest response to chemoattractive LTs. These results indicate that chemoattractant responses to LTs may be identified by utilizing the under-agarose technique and computer assisted analysis of cell orientation.