Four isotopically-labelled acetates ([1-11C], [2-11C], [1-11C](2H3) and [2-11C](2H3)acetate) were synthesized and used in positron emission tomography (PET) studies of pig myocardium. The [1-11C]acetates were synthesized by carboxylation of the appropriate 1H or 2H methyl Grignard reagents immobilized on a C2 solid phase extraction column (SPE). Purification by reverse-phase HPLC, resulted in 35-45% decay-corrected radiochemical yield with a total synthesis time of 25 min, and a radiochemical purity higher than 99%. The [2-11C]acetates were synthesized by carboxylation of 11C-labelled 1H or 2H methyl lithium. Purification as above resulted in 35-55% decay-corrected radiochemical yield with a total synthesis time of 30 min, and a radiochemical purity higher than 99%. Position-specific labelling was assessed by 13C-labelling and NMR. Multiple isotopic labelling by the combination of position-specific 11C-labelling and 2H substitution, has the potential to highlight different aspects of a complex biochemical system using a selected set of tracers in comparative PET studies. An illustration of this principle is given using acetate, where citric acid cycle metabolism results in a position-specific kinetic for the 11C-label, and deuteration opens up the possibility for the proton-abstracting processes within the citric acid cycle to be assessed.