Shape change and motility of polymorphonuclear leukocytes (PMNs) are essential for host defense and require dynamic reorganizations of microfilamentous cytoskeleton by reversible polymerizatIon of G-actin into filaments (F-actin). Although clinical disorders of actin polymerization are rare, recently described simple methodologies for assaying actin dynamics in PMNs make the technique readily applicable to clinical studies. To develop a clinically useful F-actin assay, the authors investigated the optimal preparation conditions for PMN isolation that resulted in the least in vitro cytoskeletal activation and evaluated the variability in actin dynamics in acutely and chronically infected patients. Basal and chemotactic factor- activated PMN F-actin content was measured by a previously described flow cytometric technique in fixed, permeabilized, NBDphallacidin-stained PMNs isolated by centrifugation in Percoll or Ficoll-Hypaque density gradients or by counter-current elutriation. F-actin content is expressed as mean fluorescent channel or relative fluorescence intensity. Basal F-actin in PMNs prepared from countercurrent elutriation (mean fluorescent channel = 79.0 ± 4.5, n = 6) or by Ficoll Hypaque (82.0 ± 3.5, n = 4) was significantly higher than endotoxin free, Percoll purified PMNs, whether purified in bulk (56.1 ± 7.9, n = 8) or by the small volume modification applicable to clinical studies (53.3 ± 8.7, n = 15). Basal Ficoll Hypaque purified PMNs have evidence of shape change, whereas endotoxin free, Percoll purified PMNs are smooth and round and represent the most basal cell equivalent in F-actin content to a circulating PMN. In contrast, maximal F-actin in PMNs observed 45 seconds after N-formyl-L-methionyl-L-leucyl-L-phenylalanine activation (10-9 M) was highest in Ficoll Hypaque (relative fluorescence intensity = 2.21 ± 0.30, n = 4), then countercurrent elutriation (1.77 ± 0.25, n = 6) and finally, large (1.60 ± 0.21, n = 8) or mini volume (1.64 ± 0.28, n = 12) endotoxin free, Percoll purified PMNs, suggesting that preparative regimens can activate the cytoskeleton of PMNs and may affect clinical evaluation of actin dynamics in clinical or basic science studies. With the small volume (5.0 mL) modification of the endotoxin free, Percoll technique, the authors compared actin dynamics in patients with acute and chronic infections with those of healthy volunteers. This novel technique offers unique opportunities for study of the cytoskeleton in PMNs from healthy and unhealthy patients and defines optimal conditions for clinical investigations of cytoskeletal dynamics.