Luminescence-based imaging-fiber oxygen sensors (IFOSs) were utilized for the in situ measurement of oxygen consumption from intact perfused mouse hearts. IFOSs were fabricated using a technically expedient, photoinitiated polymerization reaction whereby an oxygen-sensitive polymer matrix was immobilized in a precise location on an imaging fiber's distal face. The oxygen-sensing layer used in this work comprised a transition metal complex, Ru(Ph2phen)3(2+), entrapped in a gaspermeable photopolymerizable siloxane membrane (PS802). The transduction mechanism was based upon the oxygen collisional quenching of the ruthenium complex luminescence; detection was performed utilizing an epi-fluorescence microscope/charge coupled device imaging system. IFOS measurements from working mouse hearts were validated through concurrent, blind, ex situ blood gas analyzer (BGA) measurements. The BGA and IFOS methodologies were utilized successfully to measure oxygen concentrations in aortic and pulmonary artery perfusates from the working mouse heart before and after isoproterenol administration. Coupled with coronary-flow measurements, these data were used to calculate myocardial oxygen consumption. Regression analysis of measurements of myocardial oxygen consumption showed that there was a strong correlation between the values generated by the BGA sampling and those obtained via in situ IFOS methods. To our knowledge, this research represents the first report of in situ fiber-optic sensor monitoring of oxygen content from the intact, beating mouse heart.