The biosynthesis of the alternative oxidase protein consisting of cyanide-insensitive and salicylhydroxamic acid-sensitive respiration in Aspergillus niger was investigated. Cyanide-insensitive respiration was enhanced by addition of antimycin A to the incubation mixture, but repressed by the addition of cycloheximide, emetine, puromycin (cytosolic translation inhibitors), carbonylcyanide-m-chlorophenylhydrazone (uncoupler) and actinomycin D (transcription inhibitor). These results show that the alternative oxidase protein of A. niger was synthesized de novo in cytosol and transported in mitochondria.