We report the sequence of a cDNA clone that codes for the carboxy-terminal portion of the peroxisomal protein, acyl-CoA oxidase, from the yeast, Candida tropicalis. This is a newly identified acyl-CoA oxidase sequence, most likely a second allele of POX4. The cDNA clone was expressed by in vitro transcription followed by translation. The major product, a 43-kD protein, associated with isolated peroxisomes in an in vitro import assay. More than half of the peroxisome-associated protein was protected from added protease, implying that it was internalized within the organelle. These findings indicate that there is sufficient information in the carboxy-terminal portion of the protein to target it to peroxisomes.