Recent clinical studies of patients with sepsis have shown that the delivery of adequate oxygen alone does not necessarily result in improved organ function or survival. This study was undertaken to determine if optical spectroscopy could detect higher intracellular oxygenations in isolated, perfused guinea pig hearts that have been treated with endotoxin (lipopolysaccharide [LPS]) than in controls. Four hours after intraperitoneal injection with LPS, adult guinea pigs were anesthetized, and hearts were excised and perfused in the Langendorff manner. Six control and eight LPS-exposed guinea pigs were studied. Myoglobin oxygen saturation was determined from analysis of optical reflectance spectra acquired from the left ventricular free wall. Myoglobin saturation was significantly higher at baseline with LPS than in controls (96.0% ± 0.8% vs. 89.4% ± 1.7%, P < 0.001). At the end of 30 s of ischemia, myoglobin saturation decreased to 15% ± 1% in controls, but to only 60% ± 7% in the LPS group. Myocardial performance was determined by measured left ventricular developed pressure, which was significantly depressed in the LPS-exposed hearts relative to controls (30 ± 4 mmHg vs. 67 ± 9 mmHg, P < 0.001). Myocardial oxygen consumption, calculated from measurements of arterial and venous PO2 and coronary flow, was lower in LPS hearts relative to controls (0.199 ± 0.021 mL oxygen·min-1·g-1 vs. 0.157 ± 0.006 mL oxygen·min-1·g-1). In this model of sepsis in the perfused guinea pig heart, intracellular oxygenation was higher and oxygen consumption was lower than in controls. Cellular dysfunction seen in sepsis may be caused by compromised oxygen use rather than insufficient oxygen delivery. Optical spectroscopy has the potential to noninvasively monitor patients and their responses to therapy.