AbstractAn enzymatic method for the preparative resolution of racemic carnitine (whoseL‐isomer and its acyl‐derivatives have numerous therapeutical applications) has been developed. It is based on our finding that electriceel acetylcholinesterase hydrolyzes theD‐ but not theL‐isomer of acetylcarnitine. (Another cholinesterase tested, horse serum butyrylcholinesterase, is also stereospecific and hydrolyzes only theL‐isomer of butyrylcarnitine.) Acetylcholinesterase, covalently attached to alumina, was employed for the resolution ofD,L‐carnitine; the latter was first chemically acetylated, then stereoselectively hydrolyzed with the immobilized enzyme, and finally the acetyl‐L‐carnitine andD‐carnitine produced were separated by ion‐exchange chromatography. Gram quantities ofD,L‐carnitine