Background: Technetium 99m-HL91 is a new hypoxia imaging agent that demonstrates increased uptake in ischemic, viable myocardium. This study was performed to determine whether HL91 is taken up by nonviable myocardium. Methods: Twenty-three Krebs-Henseleit buffer-perfused, isolated rat hearts were studied. Tc-99m-HL91 300 μCi was infused over 10 minutes, followed by a 60-minute clearance. Myocardial activity was monitored by use of an NaI crystal. Four groups were studied: control (flow = 12 mL/min, n = 7), low flow (flow = 1 mL/min, n = 6), no flow/reflow (60 minutes no flow/60 minutes reflow before Tc-99m-HL91 infusion, flow = 12 mL/min, n = 5), and cyanide-treated (before Tc-99m-HL91 infusion, flow = 12 mL/min, n = 5). Injury was assessed by creatine kinase, transmission electron microscopy, and triphenyltetrazolium chloride. Results: Control (no injury) and cyanide-treated (severe injury) hearts demonstrated low uptake (6.3 ± 0.5 mean ± SEM and 5.7 ± 1.2 μCi, respectively) and low 60-minute retention (13.8% ± 2.2% and 13.7% ± 3.9%, respectively). Low-flow hearts (minimal injury) demonstrated markedly increased uptake (43.5 ± 2.8 μCi, P < .01) and increased 60-minute retention (33.2% ± 2.9%, P < .01) compared with control. No-flow/reflow hearts (moderate injury) demonstrated intermediate uptake (8.7 ± 0.5 μCi, P < .05 to control), although retention was not significantly different (18.9% ± 3.5%, P = ns). Severely and rapidly injured myocardium demonstrated Tc-99m-HL91 peak uptake and retention indistinguishable from normal. Moderately injured myocardium demonstrated uptake intermediate between severely injured and low-flow-induced ischemic, viable myocardium. Conclusion: Thus Tc-99m-HL91 is not taken up or retained in nonviable and irreversibly injured myocardium.