Treatment of pulmonary hypertension with inhaled nitric oxide(NO) and hyperoxia in combination exposes all cells lining the airway to potentially toxic effects. Because NO has been shown to have both toxic and protective effects in alveolar type II cells, we conducted the present study to determine the effects of NO gas with hyperoxia on non-alveolar, epithelial cells.Methods: Small airway epithelial cells (SAEC; doubling time=32hrs) are a heterogeneous group of cells obtained from normal, distal conducting airways. Subconfluent (60%) monolayers were exposed to 95%O2+5%CO2 alone or in combination with 8ppm or 80ppm of NO. Exposure was maintained for 72hrs with testing performed at Time 0, 24hrs, 48hrs and 72hrs. Proliferation was assessed with nuclear [3H]thymidine incorporation, direct cell counts, Alamar blue(AB) and MTS. AB and MTS are absorbance assays using mitochondrial conversion of a color change indicator to assess viability and proliferation. The effects of 95%O2 with and without NO were analyzed by ANOVA with Newman-Keuls multiple comparison testing and significance set at p<0.05. Results:[3H]thymidine decreased tenfold in all groups (ANOVA, p<0.001) and were not affected by NO at 8ppm or 80ppm. Cell counts for all groups showed an increase at 24hrs followed by a decrease over the next 48hrs. 95%O2 had a more rapid decline in counts than either NO group (p<0.05). AB absorbance was greater in 80ppm than either 95%O2 or 8ppm at all times(p<0.05). MTS absorbance readings were greater in 80ppm than either 95%O2 or 8ppm at all times (p<0.05) while 8ppm readings were greater than 95%O2 readings at 48 and 72hrs (p<0.01). Conclusions: These results indicate that 95%O2 inhibits the growth of SAEC and that the addition of NO neither potentiates nor attenuates this response. The absorbance data suggests that NO may alter the toxic effects of O2 on mitochondrial function. The mechanism(s) producing this alteration and it's clinical implications remain to be determined.