The mature form of phytanoyl-coenzyme A 2-hydroxylase (PAHX), a nonheme Fe(II)- and 2-oxoglutarate-dependent oxygenase, catalyzes the alpha-hydroxylation of phytanoyl-CoA within peroxisomes. Mutations in PAHX result in some forms of adult Refsum's disease. Unprocessed PAHX (pro-PAHX) contains an N-terminal peroxisomal targeting sequence that is cleaved to give mature PAHX (mat-PAHX). Previous studies have implied a difference in the substrate specificity of the unprocessed and mature forms of PAHX. We demonstrate that both forms are able to hydroxylate a range of CoA derivatives, but under the same assay conditions, the N-terminal hexa-His-tagged unprocessed form is less active than the nontagged mature form. Analyses of the assay conditions suggest a rationale for the lack of activity previously reported for some substrates (e.g. isovaleryl-CoA) for the (His)6pro-PAHX. Site-directed mutagenesis was used to support proposals for the identity of the iron binding ligands (His-175, Asp-177, His-264) of the 2-His-1-carboxylate motif of PAHX. Mutation of other histidine residues (His-213, His-220, His-259) suggested that these residues were not involved in Fe(II) binding.