Several reports during the last three decades have indicated that biosynthesis of N-acetylaspartate (NAA) occurs primarily in the mitochondria. But a recent report by Lu et al. in this journal [2004; 122: 71-78] and subsequent two reports that cited those data suggested a predominant microsomal localization of the NAA biosynthetic enzyme, which is surprising in view of what is known about the biological functions of NAA. Therefore we reinvestigated this issue in rat brain homogenates using a similar fractionation procedure used by Lu et al. but without the loss of enzyme activity that they have encountered. We found that about 70% of the total Asp-NAT activity in the crude supernatant was present in the mitochondrial fraction which is about 5 times more than that in the microsomes. We found similar results in the case of the enzyme from bovine brain. In subsequent studies, we also have found that Asp-NAT activity in the bovine brain is very similar to that in the rat brain in substrate specificity and chromatographic characteristics including the high molecular weight pattern (approx. 670 kD) on size-exclusion HPLC.