A histochemical procedure has been developed for staining aminoacylase-1 (ACY-1) isozymes after electrophoresis on cellulose acetate membranes. N-Formyl-L-methionine and N-acetyl-L-methionine were excellent enzyme substrates in the staining reaction. The ACY-1 isozymes from tissue culture cells of several vertebrate species showed distinguishable electrophoretic patterns. The ACY-1 isozymes in extracts of mouse, human, owl monkey, and African green monkey kidney cells each had electrophoretic mobilities different from those of peptidases S, A, and C from the same cells. Exept for African green monkey kidney (CV-1) cells, the animal species expressed a single anodally migrating ACY-1 band. Human-mouse somatic cell hybrids containing the short arm of human chromosome 3 expressed three ACY-1 bands, as would be predicted from the dimeric structure of the enzyme. CV-1 cells expressed two (or three) ACY-1 bands, suggesting the possibility that CV-1 cells contained two alleles at a single locus or two genetic loci for ACY-1.