Electron paramagnetic resonance (EPR) studies were conducted to examine oxygen radical generation following PMN activation by N-formyl-1-methionyl-1-leucyl-1-phenylalanine (fMLP) in the presence or absence of phalloidin and cytochalasin B (CB), agents which stabilize or disrupt f-actin, or taxol and colchicine which stabilize and disrupt microtubule cytoskeletal structures respectively. PMN oxyradical production was monitored using the spin trap 5,5-dimethyl-1-pyrroline n-oxide (DMPO). PMN when unstimulated, treated with phalloidin (10 −6–10 −8M), CB (10 −6–10 −8M), taxol (10 −6–10 −8M), or colchicine (10 −6–10 −8M), did not produce a detectable DMPO signal. Stimulation with fMLP (10 −6M), however, resulted in a significant hydroxyl radical signal which was augmented by PMN treatment with CB (10 −6–10 −7M, p < 0.05) and attenuated following PMN treatment with phalloidin (10 −6–10 −7M, p < 0.05). Interestingly, colchicine treatment (10 −6–10 −8M) significantly attenuated fMLP-mediated oxyradical production, whereas taxol (10 −6–10 −7M) significantly increased PMN oxyradical production. These data suggest that stabilization of f-actin and disruption of microtubules attenuates the PMN oxidative burst, whereas disruption of f-actin and stabilization of microtubules increases radical production. These findings suggest cytoskeletal domain-specific contributions to PMN oxidative activity.