In the work described here we were able to confirm the Toceranib annotation of the C. reinhardtii ERG3 ortholog as a C-5 sterol desaturase by both phenotypic complementation and direct biochemical analysis. While the C. reinhardtii ERG3 ORF does function in yeast, it must be noted that it does not complement as fully as the yeast ERG3 gene. There are several explanations for this. Traditionally, 2 micron plasmids have been used in yeast for heterologous complementation, in order to increase the copy number of the plasmid and possibly circumvent issues regarding variations in protein stability, codon usage, and enzyme activity across species. This study uses a moderate copy number plasmid. Secondly, yeast and C. reinhardtii undergo two very different biochemical pathways for the production of sterols. Yeast use the traditional MVA pathway for the synthesis of sterols, while C. reinhardtii utilizes the DOXP pathway for the synthesis of sterols. Evolutionary differences across species allow for several variations in the ergosterol biosynthetic pathway. For example, C. reinhardtii and S. cerevisiae produce precursors to ergosterol by two independent pathways so therefore similar enzymes can often catalyze somewhat different reactions. Finally, ergosterol biosynthesis is a very metabolically taxing process that requires molecular oxygen, sources of energy, and optimal temperatures. Despite these potential complications, complementing the yeast ERG3 null mutation with the ERG3 gene from C. reinhardtii results in the production of ergosterol, and increased survival of the cells during exposure to cycloheximide and growth on acetate. Sterol biosynthesis is very intricate in APD597 nature, and while several ERG3 orthologs from different eukaryotic species have been identified and characterized, ERG3 mutants in Chlamydomonas have not been created. Previous studies using random mutagenesis in Chlamydomonas identified mutants in the last two steps of ergosterol biosynthesis but not in the earlier steps of sterol biosynthesis, which includes ERG3.