We developed an improved quantitative method to measure in vitro polymorphonuclear leukocyte (PMN) migration using an assembly consisting of a 96-well chamber, polycarbonate filter membrane, and a 96-well microtiter plate. The convenience in setup and counting of migrated cells using this method allows processing of 80 samples and 16 controls in a short assay time of only 2 h. The peroxidase contained in PMNs was used as a marker enzyme to determine the number of migrated cells. Peroxidase released from lysed migrated cells was detected with an enzymatic method utilizing o-dianisidine as substrate. Photometric measurement was performed with a conventional microtiter plate reader at a wavelength of 405 nm. Optical density readings obtained using the enzymatic assay correlated with the number of migrated cells in a linear fashion up to 1 x 10(5) cells/well. The sensitivity of the enzymatic assay was sufficient to determine cell counts as low as 500 PMNs. PMNs lost no measurable amounts of peroxidase during the migration assay when ZAS was used as the chemoattractant. A calibration method was developed to make corrections for variations in the peroxidase content of different cell preparations and changes in the peroxidase content of cells exposed to the chemoattractant. High speed, convenient handling, and the use of standard laboratory equipment result in low cost per assay and make this migration assay ideally suited to research and clinical applications.