Cotton (Gossypium hirsutum L.) and kenaf (Hibiscus cannabinus L.) are both in the Malvaceae family but kenaf is significantly more resistant to the plant pathogen Verticillium dahliae than is cotton. When infected by pathogens, both plants produce secondary metabolites termed phytoalexins that are toxic to the pathogen. We identified a phytoalexin in kenaf, o-hibiscanone, which is ten times more toxic to V. dahliae than the most potent phytoalexin produced by cotton. If the genes that control the biosynthesis of o-hibiscanone were introduced and expressed in cotton, we expect that resistance to this pathogen would increase. For successful expression in cotton, it is probably necessary that key biosynthetic intermediates in both plants have the same absolute stereochemistry. One of the biosynthetic intermediates that occurs in both plants is 3-hydroxy-a-calacorene. The compound is produced in the plants in very small quantities. Thus, neither the relative nor the absolute configuration of 3-hydroxy-a-calacorene had been determined. However, the florets of Heterotheca inuloides Cass. had been reported to contain significant quantities of (-)-3-hydroxy-a-calacorene. We were therefore able to isolate milligram quantities of the compound from H. inuloides. We submitted the crystalline compound to X-ray crystallographic analysis. The X-ray analysis revealed that the absolute configuration of the compound from H. inuloides was (8R)-(-)-3-hydroxy-a-calacorene. To decipher the relative configurations of the compound in H. inuloides versus cotton versus kenaf, we utilized an HPLC method using a chiral stationary phase column to separate the enantiomers. Comparison of the chromatograms from isolated quantities (minute in the case of cotton and kenaf) of 3-hydroxy-a-calacorene from the three plant types with those of synthetic racemic 3-hydroxy-a-calacorene established that the configuration of (-)-3-hydroxy-a-calacorene in H. inuloides was opposite of that in cotton and kenaf. Importantly, the absolute configuration of this key biosynthetic phytoalexin precursor is the same in both cotton and kenaf. Thus, we expect that the transfer and expression of appropriate kenaf genes in cotton would enhance the potency of the phytoalexin mix in the latter.