Peroxisomes play a major role in human cellular lipid metabolism, including the β‐oxidation of fatty acids. The most frequent peroxisomal disorder is X‐linked adrenoleukodystrophy (X‐ALD), which is caused by mutations in the ABCD1 gene. The protein involved, called ABCD1, or alternatively ALDP, is a member of the ATP‐binding‐cassette (ABC) transporter family and is located in the peroxisomal membrane. The biochemical hallmark of X‐ALD is the accumulation of very long‐chain fatty acids (VLCFAs), due to an im paired peroxisomal β‐oxidation. Although this suggests a role of ALDP in VLCFA import, no experimental evidence is available to substantiate this. In the yeast Saccharomyces cerevisiae, peroxisomes are the exclusive site of fatty acid β‐oxidation. Earlier work has shown that uptake of fatty acids into peroxisomes may occur via two routes, either as free fatty acids thus requiring intraperoxisomal activation into acyl‐CoA esters or as long‐chain acyl‐CoA esters. The latter route involves the two peroxisomal half ABC transporters Pxalp and Pxa2p that form a heterodimeric complex in the perox isomal membrane. Using different strategies, including the analysis of intracellular acyl‐CoA esters by tandem‐MS, we show that the Pxa1p/Pxa2p heterodimer is involved in the transport of a spectrum of acyl‐CoA esters. Interestingly, we found that the mutant phenotype of the pxa1/pxa2Δ mutant can be rescued, at least par tially, by the sole expression of the human ABCD1 cDNA coding for ALDP, the protein that is defective in the human disease X‐linked adrenoleukodystrophy. Our data indicate that ALDP can function as a ho modimer and is involved in the transport of acyl‐CoA esters across the peroxisomal membrane.— van Roer mund, C. W. T., Visser, W. F., IJlst, L., van Cruchten, A., Boek, M., Kulik, W., Waterham, H. R., Wanders, R. J. A. The human peroxisomal ABC half transporter ALDP functions as a homodimer and accepts acyl–CoA esters. FASEB J. 22, 4201–4208 (2008)