AbstractCopper-64-Diacetyl-bis(N⁴-methylthiosemicarbazone) [⁶⁴Cu][Cu(ATSM)] is a hypoxia-targeting PET tracer with applications in oncology and cardiology. Upon entering a hypoxic cell, [⁶⁴Cu][Cu(II)(ATSM)] is reduced to a putative [⁶⁴Cu][Cu(I)(ATSM)]⁻ species which dissociates to deposit radiocopper, thereby providing hypoxic contrast. This process may be dependent upon protonation arising from intracellular acidosis. Since acidosis is a hallmark of ischemic tissue and tumors, the hypoxia specificity of [⁶⁴Cu][Cu(ATSM)] may be confounded by changes in intracellular pH. We have therefore determined the influence of intracellular pH on [⁶⁴Cu][Cu(ATSM)] pharmacokinetics. Using isolated perfused rat hearts, acidosis was induced using an ammonium pre-pulse method, with and without hypoxic buffer perfusion. Cardiac [⁶⁴Cu][Cu(ATSM)] pharmacokinetics were determined using NaI detectors, with intracellular pH and cardiac energetics monitored in parallel by ³¹P NMR. To distinguish direct acidotic effects on tracer pharmacokinetics from acidosis-induced hypocontractility, parallel studies used lidocaine perfusion to abolish cardiac contraction. Hypoxic myocardium trapped [⁶⁴Cu][Cu(ATSM)] despite no evidence of it being acidotic when characterised by ³¹P NMR. Independent induction of tissue acidosis had no direct effect on [⁶⁴Cu][Cu(ATSM)] pharmacokinetics in either normoxic or hypoxic hearts, beyond decreasing cardiac oxygen consumption to alleviate hypoxia and decrease tracer retention, leading us to conclude that tissue acidosis does not mediate the hypoxia selectivity of [⁶⁴Cu][Cu(ATSM)].