Inosine, a naturally occurring purine with anti-inflammatory properties, was assessed as a possible modulator of hyperoxic damage to the pulmonary alveolar epithelium. Rats were treated with IP inosine, 200mg/kg twice daily during 48h exposure to >90% oxygen. The alveolar epithelial type 2 cells (AEC2) were then isolated and cultured. AEC2 isolated from inosinetreated hyperoxic rats had less DNA damage and had increased anti-oxidant status, compared with AEC2 from hyperoxic rats. Inosine treatment during hyperoxia also reduced the proportion of AEC2 in S and G2/M phases of the cell cycle and increased levels of the DNA repair enzyme OGG. BAL recovered from hyperoxic, inosine-treated rats contained 3 fold higher levels of active TGF- than BAL from rats exposed to hyperoxia alone, and Smad2 was activated in AEC2 isolated from these animals. ERK1/2 was activated both in freshly isolated and 24hcultured AEC2 by in vivo inosine treatment, while blockade of the MAPK pathway in vitro reduced the protective effect of in the vivo inosine treatment. Taken together, the data suggest that inosine treatment during hyperoxic exposure results in protective signaling mediated through pathways downstream of MEK. Thus inosine may deserve further evaluation for its potential to reduce hyperoxic damage to the pulmonary alveolar epithelium.