The fact that thiamine pyrophosphate is synthesized in cytosol necessitates its uptake into mitochondria. The ability of mitochondria to take up externally added thiamine pyrophosphate was investigated by measuring the intramitochondrial thiamine pyrophosphate content using an enzymatic method. Thiamine pyrophosphate uptake by isolated rat liver mitochondria was found to occur in a time- and temperature-dependent manner. Uptake shows saturation characteristics with Km and Vmax values equal to about 20 microM and 700 pmol/min x mg protein, respectively, and is inhibited by certain nonpenetrating compounds. The inhibition of thiamine uptake by thiamine pyrophosphate and the efflux of endogenous thiamine pyrophosphate, caused by externally added thiamine, suggest the existence of a thiamine pyrophosphate/thiamine antiporter which could play an active role in the turnover of intramitochondrial thiamine pyrophosphate linked enzymes.