Alveolar macrophages (AM) may be exposed to a range of CO₂ and pH levels depending on their location in the alveoli and the health of the lung. Cytokines produced by AM contribute to inflammation in acute lung injury (ALI). Current ventilatory practices for the management of ALI favour low tidal volumes, which can give rise to increases in CO₂ and changes in pH of the alveolar microenvironment. Here we examined the effect of CO₂ on cytokine release from LPS-stimulated rat AM. AM were incubated for 1-4 h under different atmospheric gas mixtures ranging from 2.5-20% CO₂. To distinguish between effects of pH and CO₂, the culture media was also buffered to pH 7.2 with NaHCO₃. Cell metabolic activity, but not cell viability, decreased and increased significantly after 4 h at 20% and 2.5% CO₂, respectively. Increasing CO₂ decreased TNF- secretion but had no effect on lysate TNF-. Buffering the media abated the effects of CO₂ on TNF- secretion. CO₂ increased CINC-1 secretion only when the pH was buffered to 7.2. Effects of CO₂ on cytokine responses were reversible. In conclusion, the effects of CO₂ on cytokine lysate levels and/or secretion in AM are cytokine specific and depending on both the cytokine and the immediate microenvironment, may be beneficial or detrimental to ALI.