The Zygomycetes fungus Mortierella alpina was cultured to growth arrest to assess the phytosterol biosynthesis pathway in a less-advanced fungus. The mycelium was found to produce 13 sterols, but no ergosterol. The sterol fractions were purified to homogeneity by HPLC and their identifies determined by a combination of GC–MS and 1 H NMR spectroscopy. The principal sterol of the mycelium was cholesta-5, 24-dienol (desmosterol) (83%), with lesser amounts of 24β-methyl-cholesta-5,25(27)-dienol (codisterol) (2%), 24-methyldesmosterol (6%), 24(28)-methylene cholesterol (3%) and lanosterol (3%) and several other minor compounds (3%). The total sterol accounted for approximately 0.07% of the mycelial dry wt. Mycelium fed methionine-methyl- 2 H 3 for 6 days, generated 3 2 H-24-methyl(ene) sterols, [C28- 2 H 2 ]24(28)-methylenecholesterol, [C28- 2 H 3 ]24-methylcholesta-5,24-dienol and [C28- 2 H 3 ]24β-methyl-cholesta-5,25(27)-dienol. The formation of the 24-methyl sterols seems to be catalyzed by the direct methylation of a common Δ 24 -acceptor sterol thereby bypassing the intermediacy of an isomerization step for rearrangement of the Δ 24(28) -bond to Δ 25(25) -position as operates in Ascomycetes fungi and all plants.