Mammalian aminoacylase-1 (Acy1) participates in the breakdown of N-acetylated amino acids during intracellular protein catabolism. Acy1is most abundantly expressed in the kidney tubular epithelium. Lately, Acy1 deficiency was identified in children with increased urinary excretionof several N-acetylamino acids. Here we report detailed N-acetylamino acid specificity profiles for human and porcine Acy1 based on steadystate kinetic measurements. We found that LLC-PK1 cells, a model of the porcine kidney proximal tubular epithelium, robustly express Acy1.For the first time, we demonstrate uptake and utilization of N-acteylleucine and -methionine in replacement of the free amino acid, respectively,in cultured epithelial cells. Our data are consistent with a specific role of kidney Acy1 in the salvage of amino acids originating from systemicdegradation of N-acetylated proteins.