The alveolar macrophage generation of interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) cytokines has been implicated in the recruitment of neutrophils into acutely injured lungs. To examine the role of these cytokines in neutrophil chemotaxis, cytokine mRNA transcripts and content were examined in macrophages lavaged from rats immediately following 6 hr exposure to air or 1 ppm ozone. Ozone exposure enhanced the number of lavaged macrophages demonstrating mRNA transcripts and immunocytochemical staining for IL-1 beta and TNF-alpha. These changes occurred prior to ozone-induced increases in permeability and lavageable neutrophils. The supernatant from in vitro macrophage cultures demonstrated ozone-associated enhancements in neutrophil chemotactic activity and in IL-1 beta and TNF-alpha levels. However, treatment of the macrophage-conditioned media with anti-IL-1 beta and anti-TNF-alpha antibodies separately and in combination demonstrated that these cytokines were not directly responsible for the observed neutrophil chemoattraction. However, coculturing the macrophages with anti-IL-1 beta and anti-TNF-alpha together, but not separately, resulted in a 44% inhibition of media chemotactic activity, suggesting that maximal macrophage generation of chemoattractants was dependent on either IL-1 beta or TNF-alpha. The mRNA transcripts for the neutrophil chemoattractants macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC) were found to be enhanced in cultured macrophages from ozone-exposed rats, but reduced on incubation with anti-IL-1 beta and anti-TNF-alpha together. These results demonstrated that ozone-induced enhancements in IL-1 beta and TNF-alpha productions appear not to be associated directly with neutrophil chemoattraction, but are more likely involved in stimulating the generation of the neutrophil chemoattractants MIP-2 and CINC.