In plants, the conversion of cycloartenol into functional phytosterols requires the removal of the two methyl groups at C-4 by an enzymic complex including a sterol 4α-methyl oxidase (SMO). We report the cloning of candidate genes for SMOs in Arabidopsis thaliana, belonging to two distinct families termed SMO1 and SMO2 and containing three and two isoforms respectively. SMO1 and SMO2 shared low sequence identity with each other and were orthologous to the ERG25 gene from Saccharomyces cerevisiae which encodes the SMO. The plant SMO amino acid sequences possess all the three histidine-rich motifs (HX₃H, HX₂HH and HX₂HH), characteristic of the small family of membrane-bound non-haem iron oxygenases that are involved in lipid oxidation. To elucidate the precise functions of SMO1 and SMO2 gene families, we have reduced their expression by using a VIGS (virus-induced gene silencing) approach in Nicotiana benthamiana. SMO1 and SMO2 cDNA fragments were inserted into a viral vector and N. benthamiana inoculated with the viral transcripts. After silencing with SMO1, a substantial accumulation of 4,4-dimethyl-9β,19-cyclopropylsterols (i.e. 24-methylenecycloartanol) was obtained, whereas qualitative and quantitative levels of 4α-methylsterols were not affected. In the case of silencing with SMO2, a large accumulation of 4α-methyl-Δ⁷-sterols (i.e. 24-ethylidenelophenol and 24-ethyllophenol) was found, with no change in the levels of 4,4-dimethylsterols. These clear and distinct biochemical phenotypes demonstrate that, in contrast with animals and fungi, in photosynthetic eukaryotes, these two novel families of cDNAs are coding two distinct types of C-4-methylsterol oxidases controlling the level of 4,4-dimethylsterol and 4α-methylsterol precursors respectively.