The biosynthesis of wax components containing secondary functional groups was investigated in the current study. Two fundamentally different pathways were proposed to introduce the secondary functional groups. One pathway involves hydroxylation of elongated substrates. Wax components characterized by two functional groups located on or near the centre of the carbon chain, nonacosane-14,15-diol, -14,16-diol and -13,15-diol as well as corresponding ketols were identified for the first time in Arabidopsis stem wax. The alkanediols and ketols were dominated by the C-14,15 isomers. The absence of alkanediols and ketols in Arabidopsis mah1 mutants that are deficient in secondary alcohol biosynthesis confirmed the biosynthetic relationship between secondary alcohols and alkanediols/ketols (Chapter 3). In pea (Pisum sativum) leaf wax, two novel compound classes were identified as primary/secondary alcohols dominated by octacosane-1,14-diol and secondary/secondary alkanediols hentriacontane-9,16-diol, -8,15-diol and -10,17-diol. Co-localization of the secondary/secondary alkanediols and hentriacontan-15-ol and -16-ol pointed to a biosynthetic relationship (Chapter 4). The diverse structures of compounds identified in the current study suggested that hydroxylases can use substrates other than alkanes. The predominance of isomers within homologues indicated a regiospecificity of the hydroxylases involved in wax biosynthesis. In addition to hydroxylation, secondary functional groups could also be introduced through elongation of carbon chains. Homologous series of 5-hydroxyaldehydes (C₂₄ and C₂₆-C₃₆) and 1,5-alkanediols (C₂₈-C₃₈) were identified in yew (Taxus baccata) needle wax. The relative position of both functional groups suggested that these two compound classes are biosynthetically related and their secondary functional groups are introduced during elongation (Chapter 5). The results of incubation of ¹⁴C-labeled malonyl-CoA and acyl-CoAs with different chain lengths in the presence of California poppy (Eschscholzia californica) microsomes provided the first evidence to support the elongation hypothesis. The results indicated that a carbonyl group rather than a hydroxyl group is introduced during elongation. To provide molecular tools for further investigations of the hypothetical pathway, three full length cDNAs encoding putative KCSs were cloned and one of them, PKCSI, was functionally characterized.