We have developed a new single photon emission computerized tomography (SPECT) hotspot quantification method incorporating extra cardiac activity correction and hotspot normal limit estimation. The method was validated for estimation accuracy of myocardial tracer focal uptake in a chronic canine model of myocardial infarction (MI). Dogs (n = 4) at 2 weeks post MI were injected with Tl-201 and a Tc-99m-labeled hotspot tracer targeted at matrix metalloproteinases (MMPs). An external point source filled with Tc-99m was used for a reference of absolute radioactivity. Dual-isotope (Tc-99m/T1-201) SPECT images were acquired simultaneously followed by an X-ray CT acquisition. Dogs were sacrificed after imaging for myocardial gamma well counting. Images were reconstructed with CT-based attenuation correction (AC) and without AC (NAC) and were quantified using our quantification method. Normal limits for myocardial hotspot uptake were estimated based on 3 different schemes: maximum entropy, meansquaredor minimization (MSEM) and global minimization. Absolute myocardial hotspot uptake was quantified from SPECT images using the normal limits and compared with well-counted radioactivity on a segment-by-segment basis (n = 12 segments/dog). Radioactivity was expressed as % injected dose (%ID). There was an excellent correlation (r = 0.78-0.92) between the estimated activity (%ID) derived using the SPECT quantitative approach and well-counting, independent of AC. However, SPECT quantification without AC resulted in the significant underestimation of radioactivity. Quantification using SPECT with AC and the MSEM normal limit yielded the best results compared with well-counting. In conclusion, focal myocardial "hotspot" uptake of a targeted radiotracer can be accurately quantified in vivo using a method that incorporates SPECT imaging with AC, an external reference, background scatter compensation, and a suitable normal limit. This hybrid SPECT/CT approach allows for the serial non-invasive quantitative evaluation of molecular targeted tracers in the heart.