Aims: Isoprene (2-methyl-1,3-butadiene; C5H8) is naturally produced by photosynthesis and emitted in the atmosphere by the leaves of many herbaceous, deciduous and woody plants. Fermentative yeast and fungi (Ascomycota) are not genetically endowed with the isoprene production process. The work investigated whether Ascomycota can be genetically modified and endowed with the property of constitutive isoprene production. Methods and Results: Two different strategies for expression of the IspS gene in Saccharomyces cerevisiae were employed: (i) optimization of codon usage of the IspS gene for specific expression in S.cerevisiae and (ii) multiple independent integrations of the IspS gene in the rDNA loci of the yeast genome. Copy number analysis showed that IspS transgenes were on the average incorporated within about 25% of the endogenous rDNA. Codon use optimization of the Pueraria montana (kudzu vine) IspS gene (SckIspS) for S.cerevisiae showed fivefold greater expression of the IspS protein compared with that of nonoptimized IspS (kIspS). With the strategies mentioned earlier, heterologous expression of the kudzu isoprene synthase gene (kIspS) in S.cerevisiae was tested for stability and as a potential platform of fermentative isoprene production. The multi-copy IspS transgenes were stably integrated and expressed for over 100 generations of yeast cell growth and constitutively produced volatile isoprene hydrocarbons. Secondary chemical modification of isoprene to a number of hydroxylated isoprene derivatives in the sealed reactor was also observed. Conclusion: Transformation of S.cerevisiae with the Pueraria montana var. lobata (kudzu vine) isoprene synthase gene (IspS) conferred to the yeast cells constitutive isoprene hydrocarbons production in the absence of adverse or toxic effects. Significance and Impact of the Study: First-time demonstration of constitutive isoprene hydrocarbons production in a fermentative eukaryote operated through the mevalonic acid pathway. The work provides concept validation for the utilization of S.cerevisiae, as a platform for the production of volatile hydrocarbon biofuels and chemicals.