Arabidopsis wax components containing secondary functional groups were examined (i) to test the biosynthetic relationship between secondary alcohols and ketols and (ii) to determine the regiospecificity and substrate preference of the enzyme involved in ketol biosynthesis. The stem wax of Arabidopsis wild type contained homologous series of C(27) to C(31) secondary alcohols (2.4 microg cm(-2)) and C(28) to C(30) ketones (6.0 microg cm(-2)) dominated by C(29) homologues. In addition, compound classes containing two secondary functional groups were identified as C(29) diols (approximately 0.05 microg cm(-2)) and ketols (approximately 0.16 microg cm(-2)). All four compound classes showed characteristic isomer distributions, with functional groups located between C-14 and C-16. In the mah1 mutant stem wax, diols and ketols could not be detected, while the amounts of secondary alcohols and ketones were drastically reduced. In two MAH1-overexpressing lines, equal amounts of C(29) and C(31) secondary alcohols were detected. Based on the comparison of homologue and isomer compositions between the different genotypes, it can be concluded that biosynthetic pathways lead from alkanes to secondary alcohols, and via ketones or diols to ketols. It seems plausible that MAH1 is the hydroxylase enzyme involved in all these conversions in Arabidopsis thaliana.