Branched-chain fatty acids and related molecules are important components of the human diet and are also used as drug molecules. The presence of a 2-methyl group on the carbon chain prevents ‘normal’ beta-oxidation in mitochondria, and these compounds are (initially) metabolised in peroxisomes. Important examples include bile acids (derived from cholesterol), phytanic acid (a dietary source of 2-methyl acids), and ibuprofen. These acids can exist as either the (R)- or (S)- isomers, but beta-oxidation is only possible for the (2S)-isomers. Alpha-methylacyl-CoA racemase (AMACR) catalyses chiral inversion of these acids (as their CoA esters), and thus regulates entry of metabolites into the beta-oxidation pathway. AMACR 1A is over-expressed in prostate, breast, colon and other cancers and is used as a prostate cancer marker, and several other splice variants are over-expressed in prostate cancer in addition to AMACR 1A. Reducing expression of AMACR 1A prevents proliferation of prostate cancer cells, suggesting it is a novel anti-cancer drug target. Despite this importance little is known about the biochemistry of the enzyme. Human AMACR 1A was over-expressed in E. coli using the pET30 Xa vector and purified on around a 20 mg scale by metal-chelate chromatography. A chiral synthesis of substrate was developed from decanoic acid. The protected acid was specifically methylated using Evan’s chemistry before deprotection and conversion to the CoA ester. Enzyme assays derivatised the CoA product mixture followed by gas chromatographic analysis. AMACR 1A can catalyse chiral inversion of the synthetic substrate 2-methyldecanoyl-CoA in both directions with an equilibrium ratio (2R/2S) of ~ 2:1. The enzyme has been characterised with respect to its catalytic properties. The results will facilitate biochemical studies on this important enzyme and its development as a drug target.