We developed a nonradioactive assay to measure spermidine/spermine N(1)-acetyltransferase (SSAT) activity by high-performance liquid chromatography (HPLC). N(1)-dansylnorspermine was prepared and evaluated as a substrate of acetylation with acetyl-CoA by SSAT in rat hepatoma (HTC) cells. Kinetic studies revealed that the K(m) values of N(1)-dansylnorspermine and acetyl-CoA were approximately 11 and 13 microM, respectively. When the assay method was applied to HTC cell samples, the SSAT activity, even at the control level, could easily be detected in as few as 20 microg protein of cell extract corresponding to 1 x 10(5) cells per determination, and 100 samples could be analyzed overnight. Thus, our HPLC method is a rapid and sensitive assay for the measurement of SSAT activity.