Subcellular fractions of rat liver were obtained by differential centrifugation. The fractions enriched in lysosomes or microsomes were further fractionated in discontinuous sucrose density gradients or continuous iso-osmotic gradients made of modified colloidal silica. The fractions were analyzed for marker enzymes of the different subcellular organelles and for acetyl-CoA: alpha-glucosaminide N-acetyltransferase. The acetyl-CoA: alpha-glucosaminide N-acetyltransferase activity showed a bimodal distribution. About one fourth of the activity was associated with lysosomes, whereas the greater part of the activity was recovered in the microsomal fraction. Plasma membrane-enriched fractions contained only trace amounts of acetyl-CoA: alpha-glucosaminide N-acetyltransferase. The lysosomal and microsomal acetyl-CoA: alpha-glucosaminide N-acetyltransferases are membrane-bound and can be solubilized with Triton X-100. The pH dependence and sensitivity to various ions was similar for the lysosomal and microsomal enzyme.