Fatty acid β-oxidation in peroxisomes requires the continued uptake of fatty acids or their derivatives into peroxisomes and export of β-oxidation products plus oxidation of NADH to NAD. In an earlier study we provided evidence for the existence of an NAD(H) redox shuttle in which peroxisomal malate dehydrogenase plays a pivotal role. In analogy to the NAD(H)-redox-shuttle systems in mitochondria we have investigated whether a malate/aspartate shuttle is operative in peroxisomes. The results described in this paper show that peroxisomes of oleate-grown Saccharomyces cerevisiae contain aspartate aminotransferase (AAT) activity. Whereas virtually all cellular AAT activity was peroxisomal in oleate-grown cells, we found that in glucose-grown cells most of the AAT activity resided in the cytosol. We demonstrate that the gene AAT2 codes for the cytosolic and peroxisomal AAT activities. Disruption of the AAT2 gene did not affect growth on oleate. Furthermore β-oxidation of palmitate was normal. These results indicate that AAT2 is not essential for the peroxisomal NAD(H) redox shuttle.