Enzymes from thermophiles are preferred for industrial applications because they generally show improved tolerance to temperature, pressure, solvents, and pH as compared with enzymes from mesophiles. However, nearly all thermostable enzymes used in industrial applications or available commercially are produced as recombinant enzymes in mesophiles, typically Escherichia coli. The development of high-temperature bioprocesses, particularly those involving cofactor-requiring enzymes and/or multi-step enzymatic pathways, requires a thermophilic host. The extreme thermophile most amenable to genetic manipulation is Thermus thermophilus, but the study of expression of heterologous genes in T. thermophilus is in its infancy. While several heterologous genes have previously been expressed in T. thermophilus, the data reported here include the first examples of the functional expression of a gene from an archaeal hyperthermophile ( bglA from Pyrococcus woesei), a cofactor-requiring enzyme ( dszC from Rhodococcus erythropolis IGTS8), and a two-component enzyme ( carBa and carBb from Sphingomonas sp. GTIN11). A thermostable derivative of pnbA from Bacillus subtilis was also expressed, further expanding the list of genes from heterologous hosts that have been expressed in T. thermophilus.