The mechanisms by which endotoxemia causes cardiac depression have not been fully elucidated. The present study examined the involvement of nitric oxide (NO) in this pathology. Rats were infused with either lipopolysaccharide (LPS) or saline, and the plasma and myocardial NO₂^-/NO₃^- (NOx) concentrations were measured before, or 3, 6, and 24 h after treatment. The hearts were then immediately isolated, mounted in a Langendorff apparatus, and the left ventricular developed pressure (LVDP) was determined prior to biochemical analysis of the myocardium. LPS injection effected the expression of inducible NO synthase (iNOS) in the myocardium, a marked increase in plasma and myocardial NOx levels, and a significant decline in LVDP, when compared to saline controls. The LPS-induced NO production and concomitant cardiac depression were most pronounced at 6 h after LPS injection, and were accompanied by a significant increase in myocardial guanosine 3', 5'-cyclic monophosphate (cGMP) content. Myocardial ATP levels were not significantly altered after LPS injection. Significant negative correlation was observed between LVDP and myocardial cGMP content, as well as between LVDP and plasma NOx levels. Aminoguanidine, an inhibitor of iNOS, significantly attenuated the LPS-induced NOx production and contractile dysfunction. Furthermore, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase, significantly decreased myocardial cGMP content and attenuated the contractile depression, although either aminoguanidine or ODQ was not able to completely reverse myocardial dysfunction. Our data suggest that endotoxin-induced contractile dysfunction in rat hearts is associated with NO production by myocardial iNOS, and a concomitant increase in myocardial cGMP.