Injection of phorbol 12-myristate 13-acetate (PMA) into polymorphonuclear leukocyte (PMN)-depleted, PMN cytoplast-repleted New Zealand White rabbits caused the development of acute lung injury in vivo. PMN cytoplasts are nucleus- and granule-free vesicles of cytoplasm capable of releasing toxic O2 radicals but incapable of releasing granule enzymes. PMN cytoplasts when activated by PMA reduced 66 ± 12.7 nmol of cytochrome c compared with 2.6 ± 07 nmol in their resting state and did not release a significant quantity of granule enzymes (P > 0.05). Injection of PMA into New Zealand White rabbits caused a significant decrease (P < 0.05) in the number of circulating cytoplasts. Increases in lung weight-to-body weight ratios in PMA-treated rabbits (9.8 ± 0.5 x 10-3) compared with saline-treated rabbits (5.3 ± 0.2 x 10-3) were also noted. Levels of angiotensin-converting enzyme in lung lavage as well as the change in alveolar-arterial O2 ratio correlated with the numbers of cytoplasts in lung lavage (P = 0.001, r = 0.84 and P = 0.0166, r = 0.73, respectively). Albumin in lung lavage increased to 1,700 ± 186 mg/ml in PMA-treated rabbits from 60 ± 30 mg/ml in saline-treated rabbits. These changes were attenuated by pretreatment of rabbits with dimethylthiourea (DMTU). In vitro, cytoplasts were able to mediate increases in endothelial monolayer permeability. This was evidenced by increases in fractional transit of albumin across endothelial monolayers when treated with PMA-activated cytoplasts (0.08 ± 0.01 to 0.28 ± 0.02). Increased numbers of activated cytoplasts were able to transit through the endothelial monolayer in response to F-Met-Leu-Phe (68 ± 8 from 12 ± 2 cytoplasts/high-power field). These findings suggest that cytoplasts produce acute lung injury in vivo and cause increases in endothelial monolayer permeability in vitro. Because cytoplasts are free of granule enzymes, release O2 radicals when activated, and because DMTU prevented these responses, a significant role for toxic O2 metabolites in the production of acute lung injury is implicated.