Human alpha-methylacyl-CoA racemase (AMACR) was overexpressed in prostate cancer compared with nonmalignant tissues. The Gene Logic Inc. BioExpress database containing Affymetrix U133 GeneChip expression profiles of 4400 human normal, benign, diseased, and tumor samples from >60 tissue types was examined to determine the specificity of AMACR mRNA expression. One particular AMACR probeset was derived from an alternatively spliced exon with 88% identity to a 521-bp sequence that spans four exons of the fumarate hydratase. The predicted protein sequence revealed a novel GLGELIL peptide shared by both proteins. Whether the mitochondrial and peroxisomal AMACR described previously are distinct products from alternatively spliced transcripts remains to be determined. The determination of the cellular location and function of the altered AMACR will be critical in the elucidation of the role of AMACR in prostate cancer diagnosis and pathogenesis.