The sterols accumulated by ergosterol deficient mutants of the genes erg6, erg2, erg3, and erg5 (formerly po11, po12, po13, and po15) have been analyzed by gas liquid chromatography. Together with pure sterols obtained from the mutants, they were characterized on SE-30, OV-17, and OV-225. The effects of molecular structure on the retention characteristics of a range of C28 ergostane sterols have been studied. The double mutants obtained by crossing the single mutants were also analyzed and their sterols identified where possible. The effects of the erg mutations on the control of sterol biosynthesis in yeast are discussed.