N-Acetylaspartate (NAA) is an abundant amino acid derivative of the central nervous system that is localized primarily in neurons and has found widespread use in clinical NMR spectroscopy (MRS) as a non-invasive indicator of neuronal survival and/or viability. Its function, although still obscure, is thought to reflect its unusual metabolic compartmentalization wherein NAA synthase occurs in the neuron and aspartoacylase, the hydrolytic enzyme that removes the acetyl moiety, occurs in myelin and glia. The NAA synthase enzyme, acetyl-CoA/l-aspartate N-acetyltransferase (ANAT), was previously shown to function in mitochondria (MIT), although other subcellular fractions were apparently not examined. In this study we confirmed its presence in MIT but also found significant activity in rat brain microsomes (MIC). The reaction mixture, consisting of [(14)C]aspartate plus acetyl-CoA in Na-phosphate buffer (pH 7), gave rise to [(14)C]NAA that was separated and quantified by TLC. Reaction rates were 29.0+/-0.46 and 6.27+/-0.27 nmol/h/mg for MIC and MIT, respectively. K(m) values and pH optima were similar, and both fractions showed modest enhancement of ANAT activity with the detergents Triton CF-54 and CHAPS. Our tentative conclusion is that ANAT is bimodally targeted to MIT and a component of MIC-likely endoplasmic reticulum. ANAT activity increased in both MIC and MIT between 29 and 60 days of age but differed thereafter in that only MIT ANAT showed a decrease after 1 year.