Xanthine oxidase (XO) is a major source of reactive oxygen species in the cardiovascular system. Enhanced XO activity in the failing myocardium has been associated with a reduction in inotropy; however, whether this association is causal remains to be established. To test this hypothesis, the effect of XO inhibition (oxypurinol, 100 μmol/l and allopurinol, 100 μmol/l) or activation (xanthine, 100 or 500 μmol/l) on cell shortening (3 Hz, 35°) was evaluated in left ventricular (LV) myocytes isolated from C56BL/6–129j mice. Similarly, LV superoxide production in the absence and presence of inhibitors of XO, NADPH oxidases (apocynin, 100 μmol/l) or nitric oxide synthases (LNAME, 1 μmol/l) was measured by lucigenin (5 μmol/l)-enhanced chemiluminescence. Oxypurinol and allopurinol significantly suppressed basal superoxide production and cell shortening (by about 20%), whereas xanthine caused a dose-dependent increase in cell shortening and superoxide production. In contrast, apocynin had no effect on superoxide release or cell shortening. Taken together, our findings indicate that superoxide production by XO exerts a tonic positive inotropic effect on murine LV myocytes, suggesting that the increase in XO activity in heart failure may be, at least in part, adaptive.