Trichothecenes are potent inhibitors of cytoplasmic protein synthesis which can affect the severity of plant diseases such as wheat head scab. While many trichothecene-producing fungi share the initial biosynthetic intermediates, Fusarium sp. are unique in the production of trichothecenes containing an oxygen function at C-3. Although the initial trichothecene and the final products have a C-3 hydroxyl group, the intermediate steps are acetylated at C-3. By using Chlamydomonas reinhardtii, a unicellular plant with a well-defined genetic system, we were able to test the proposal that trichothecenes with a C-3 hydroxyl are more toxic to plants, as well as demonstrate that C. reinhardtii is a promising plant trichothecene bioassay system. Seven pairs of trichothecenes with either a C-3 hydroxyl or C-3 acetyl group were assayed. Our results confirm that trichothecenes acetylated at C-3 were far less toxic to Chlamydomonas than those with a C-3 hydroxyl group.