A new sensitive method for measuring aspartoacylase activity in human skin fibroblasts using [3H]N-acetyl-L-aspartic acid (NAA) is described. Optimal assay conditions and kinetic parameters for enzyme activity were determined. The enzyme was found to have maximal activity at pH 8.5, and the Michaelis constant for the substrate N-acetylaspartate was 1.8-2.0 mmol/l. Aspartoacylase activity in control cultured human fibroblasts was 9.2 +/- 1.8 nmol/h per mg protein, compared with 1.1 +/- 0.2 in seven Canavan patients and 3.5 +/- 0.9 in four patients' parents. This method for determining aspartoacylase activity is advantageous to the previously described spectrophotometric method since it is rapid, more sensitive and has less nonspecific interference. It is possible that application of this technique to cultured ammniotic and chorionic villi cells may be used for prenatal diagnosis of Canavan's disease.