Enhancement of light transmission has been widely accepted as an empirical measure of cell aggregation in suspension. Several years ago, this measurement was introduced to the study of polymorphonuclear leukocyte (PMN) aggregation by adapting a hypothesis originally developed for platelets. Accordingly, an increase in light transmission is attributed to cell aggregation and a decrease in transmission below baseline level is indicative of increased cell symmetry. We tested this hypothesis for human PMNs by comparing the whole cell shape or the cells' aggregation state with the light transmission under particular experimental conditions. The PMN light response to the chemoattractant, f-Met-Leu-Phe, in the presence of low doses of aliphatic alcohols was associated with transient enhanced transmission, followed by a rapid decrease below baseline. In contrast to the platelet hypothesis, the below-baseline effect coincided with a decrease in PMN symmetry from spheres to wedge-shaped (polarized) cells. PMNs fixed mildly with various doses of formaldehyde (0.1% to 0.3%) were completely aggregated by the addition of 50 micrograms/mL phytohemagglutinin (PHA). Despite the complete aggregation of the PMNs, there was a dose-dependent inhibition of the above-baseline level transmission response by the fixative, demonstrating a clear dichotomy between aggregation and increased light transmission. However, PMN aggregation could be monitored by observing the pattern of enhanced light transmission coupled with decreased perpendicular light scattering immediately after the stirring of the cell suspensions was stopped. PMNs aggregated by PHA cleared from suspension very rapidly (t1/2 less than or equal to one minute), whether or not they were formalin-fixed. In contrast, unaggregated cells revealed constant transmission and perpendicular scattering intensities for as long as five minutes after the stirring was stopped. The clearance patterns of f-Met-Leu-Phe-stimulated PMNs initiated even at the time of maximally increased light transmission were indistinguishable from those of the unstimulated cells, indicating the absence of aggregation. The lack of correlation between light output and changes in cell shape or degree of aggregation of PMNs causes us to reject the hypothesis that attributes enhanced light transmission to PMN aggregation. We suggest that modulation of light transmission by PMNs stimulated with chemoattractants is due to changes in light output from subcellular objects.