Treadmill exercise in Thoroughbred horses of 2 min duration and increasing intensity resulted in increased formation and accumulation of acetylcarnitine in the working middle gluteal muscle. At high work intensities a plateau in acetylcarnitine formation was reached corresponding to approximately 70% of the total carnitine pool (approx. 30 mmol.kg-1 dry muscle). Formation of acetylcarnitine was mirrored by an equal fall in the free carnitine content, which stabilised, at the highest work intensities, at around 8 mmol.kg-1 dry muscle. Acetylcarnitine and carnitine reached their point of maximum change at a work intensity just below that resulting in the rapid production and accumulation of lactate and glycerol 3-phosphate. It is possible that the formation of acetylcarnitine is important in the regulation of the intramitochondrial acetyl CoA/CoA ratio; equally these changes may represent a blocking mechanism aimed at preventing the transfer of unwanted free fatty acids (as acylcarnitines) into the mitochondria at work intensities where they could contribute little to energy production.