α-Oxidation is the process by which fatty acids are degraded by one carbon unit. It is the obligatory degradation pathway for 3-methyl-branched fatty acids, such as phytanic acid, before these can enter the β-oxidation sequence because the 3-methyl-substitution renders the third step of β-oxidation impossible. Elucidation of the exact phytanic acid α-oxidation sequence was greatly facilitated by the detection of formate as one of its products in 1993. The pathway consists of an initial activation, subsequent 2-hydroxylation by a dioxygenase, splitting of the 2-hydroxy-3-methylacyl-CoA intermediate and subsequent dehydrogenation (1). We described that the carbon unit that is split off in the third step is formyl-CoA and the other product is a 2-methyl-branched aldehyde with one carbon less (1). The responsible enzyme was purified, sequenced, and characterized (2). It appeared to be a homotetrameric enzyme with a peroxisomal targeting sequence (PTS1) and it is the first thiamine pyrophosphate (TPP) dependent enzyme described in mammalian peroxisomes. As the substrate for this lyase contains a 3-methyl-branch, a 2-hydroxy-group and a CoA-ester, we wondered which of these constituents are necessary in order to be a substrate for the lyase. Substrate competition experiments with 2-hydroxyhexadecanoyl-CoA, 2-hydroxyoctadecanoyl-CoA, 2-hydroxyhexadecanoic acid, 2-hydroxy-3-methyl-hexadecanoyl-CoA, 3-methylhexadecanoyl-CoA, 3-methylhexadecanoic acid and 2-methyl- and 3-hydroxy-derivatives led to the conclusion that both the 2-hydroxy moiety and the CoA-ester function, but not the 3-methyl-substitution, are obligatory for the substrates of this enzyme. Further studies showed a subcellular distribution of lyase activity with 2-hydroxyoctadecanoyl-CoA, which concurred perfectly well with the profile seen for lyase activity with 2-hydroxy-3-methylhexadecanoyl-CoA as a substrate. Incubations of 2-hydroxyoctadecanoyl-CoA with the recombinant human lyase led to the production of formyl-CoA / formate and heptadecanal. All these data led to the conclusion that besides 2-hydroxy-3-methylacyl-CoA also 2-hydroxy long chain fatty acyl-CoAs are substrates for the lyase (3). Consequently, the previously named 2-hydroxyphytanoyl-CoA lyase (2-HPCL) is now called 2-hydroxyacyl-CoA lyase 1, abbreviated as HACL1 (4). As HACL1 is TPP-dependent, we studied the impact of thiamine deficiencies on the α-oxidation process in vivo (rats on thiamine deficient diets and treated with thiamine antimetabolites) and in cultured fibroblasts from patients with thiamine responsive megaloblastic anemia and controls. Our data pointed to an impact of thiamine deficiency on the α-oxidation process (5). The fact that HACL1 is the first peroxisomal enzyme described in mammals that is TPP-dependent, prompted us to investigate its presence and import in the peroxisome, as studies on the intraperoxisomal TPP were never carried out and no data are available about its import. We investigated the subcellular localization of this vitamin in rat liver. In addition to the already known cytosolic and mitochondrial pools, the presence of TPP in peroxisomes was established. The peroxisomal concentration pointed to the existence of a peroxisomal pool of TPP (unpublished data). How TPP enters the peroxisome remains to be explored. Peroxisomes are surrounded by a single membrane, and permeability for small molecules and solutes through so called peroxisomal pores has been shown previously, but also transporters have been described. Does TPP enter the peroxisome in its diphosphorylated form or as thiamine? In the latter case, it should be diphosphorylated in the peroxisome. Conversion of thiamine to TPP occurs by a thiamine pyrophosphokinase. This enzyme has been described in the cytosol. We reinvestigated its subcellular localization, in order to establish if it is also present in peroxisomes. However, its localization was exclusively cytosolic, without any activity in peroxisomes (unpublished data). In addition, the enzyme has no peroxisome targeting signal. On the basis of these findings we conclude that TPP as such is imported in the peroxisome.