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Rhodotorula (Rhodosporidium) toruloides is an oleaginous basidiomycete yeast with great biotechnological potential. Capable of accumulating lipid up to 76 % of its dry biomass and well suited to the metabolism of lignocellulosic hydrolysate, it is a good candidate for production of advanced biofuels as well as a host of other potential roles in industry. However, molecular genetic tools for manipulation of this yeast are lacking and its high genomic GC content can make routine cloning difficult. Agrobacterium tumefaciens-mediated transformation of R. toruloides CBS 14 was demonstrated, and plasmid vectors were developed for transformation of R. toruloides, including elements for Saccharomyces cerevisiae in-yeast assembly. In-yeast assembly is robust to the manipulation of GC-rich DNA and of large plasmids. Using these vectors and an EGFP reporter, a screen to identify inducible promoters was performed, and promoters from the genes NAR1, ICL1, CTR3, and MET16 identified. These promoters have independent induction/repression conditions and different levels and rates of induction. Minimal inducible promoters were determined, which are as small as 200 bp. As well as showing tight regulation of the EGFP marker, the NAR1 promoter was able to drive conditional rescue of a leu2 mutant strain. In parallel, as a proof of principle for production of advanced biofuels, hydrocarbon biosynthesis pathways were expressed in R. toruloides and analysed by GC-MS. After co-expression of Synechococcus elongatus fatty acyl-ACP reductase and fatty aldehyde decarbonylase, and E. coli ferredoxin and ferredoxin reductase, production of the alkane heptadecane was observed. To increase the availability of free fatty acids (FFA) for production of hydrocarbons by other pathways, Thermomyces lanuginosus lipase 2 was expressed, resulting in a 1.3-fold increase in the concentration of FFAs.