Hypoxia disturbs Ca2+ regulation and increases the intracellular Ca2+ concentration ([Ca2+]i), which may in turn activate the nitric oxide synthase (NOS) regulated by [Ca2+]i. Since nitric oxide (NO) reduces the isometric contractility of rat diaphragm in vitro, we hypothesized that NO contributes to the impaired force generation of an hypoxic diaphragm. The effects of different concentrations of the NOS inhibitor, NG-monomethyl-L-arginine (L-NMMA), the NO scavenger haemoglobin (150 μmol·l-1) and the NO donor spermine NONOate (Sp-NO; 1 mmol·l-1) were determined on isometric contractility during hypoxia [partial pressure of oxygen, PO2, about 7 kPa (about 54 mmHg)] and hyperoxia [PO2 about 83 kPa (about 639 mmHg)]. Hypoxia significantly reduced maximal twitch force (Ft), and submaximal tetanic force (30 Hz, F30) in all L-NMMA groups. A low concentration of L-NMMA (30 μmol·l-1) increased F30 but a high concentration (1,000 μmol·l-1) reduced F30 during hypoxia. The effects of L-NMMA on force generation were more pronounced during hypoxia compared to hyperoxia. Peak increases in F30 and Ft were observed at a concentration of 30 μmol·l-1 L-NMMA during hypoxia, but with 10 μmol·l-1 L-NMMA during hyperoxia. The same concentration of haemoglobin increased F30 and Ft less during hypoxia compared to hyperoxia. The Sp-NO reduced Ft, F30 and maximal tetanic force (F0) during hypoxia; these effects were abolished in the presence of haemoglobin. The Sp-NO did not alter Ft, F30 and F0 during hyperoxia. We conclude that NO plays a more prominent role during hypoxia and that NO contributes to the depression of force generation in the hypoxic rat diaphragm in vitro. This change may be related to a elevated NO generation within the hypoxic diaphragm .