14(R,S)-[18F]Fluoro-6-thia-heptadecanoic acid (FTHA) is a new radiolabeled long-chain fatty acid (LCFA) analog designed to undergo metabolic trapping subsequent to its commitment to the beta-oxidation pathway. Sulfur-substitution at the sixth carbon of FTHA causes a prolonging of myocardial clearance half-time (T 1/2 approximately 2 hr) in mice with little diminution of myocardial uptake (39.8 +/- 3.0% ID/g at 5 min). Heart-to-blood ratios were 20 +/- 6 and 82 +/- 16 at 1 and 60 min, respectively. In contrast, the 3-thia analog, 13(R,S)-[18F]-fluoro-3-thia-hexadecanoic acid, showed lower uptake and poor retention by heart. Myocardial uptake of FTHA was reduced by 81% (p less than 10(-5) and 87% (p less than 5 x 10(-4] in mice pretreated with the carnitine palmitoyltransferase I inhibitor, 2[5(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA) at 1 and 60 min, respectively. Radioanalytical studies showed the major metabolic fate of FTHA in control and POCA treated myocardium to be unidentified metabolite(s) that bind to tissue protein. Smaller amounts of 18F radioactivity were present in myocardium as complex lipids, fatty acid, and unidentified soluble metabolites. The results indicate metabolic trapping of FTHA in myocardium subsequent to its entry into the mitochondrion and encourage its further evaluation as a PET tracer of myocardial LCFA utilization.