In clinical practice, interferon-alpha (IFN-alpha) has been widely used as an antiviral, antitumor and immunomodulatory agent. However, its intravenous administration at large doses is associated with significant cardiovascular side-effects such as congestive heart failure and myocardial infarction. But the direct cardiovascular effects of IFN-alpha and the underlying mechanisms are not clear. In this study, we investigated the effects of IFN-alpha on contractility in Langendorff perfused rat hearts and isolated rat papillary muscles. The results showed that IFN-alpha induced a concentration-dependent negative inotropic effect in the isolated heart and papillary muscle. Pretreatment with L-NAME, a nitric oxide synthase inhibitor, attenuated this inotropic effect. Furthermore, in isolated papillary muscles IFN-alpha decreased the responsiveness to the beta-agonist isoproterenol, which was also attenuated by pretreatment with L-NAME. In conclusion, these results show that IFN-alpha induced a negative inotropic effect in normal and beta-adrenergic activated cardiac muscle at least partly via nitric oxide (NO).