We report here a novel selectable marker for the hyperthermophilic crenarchaeon Sulfolobus islandicus. The marker cassette is composed of the sac7d promoter and the hmg gene coding for the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (P(sac7d)-hmg), which confers simvastatin resistance to this crenarchaeon. The basic plasmid vector pSSR was constructed by substituting the pyrEF gene of the expression vector pSeSD for P(sac7d)-hmg with which the Sulfolobus expression plasmids pSSRlacS, pSSRAherA, and pSSRNherA were constructed. Characterization of Sulfolobus transformants carrying pSSRlacS indicated that the plasmid was properly maintained under selection. High-level expression of the His(6)-tagged HerA helicase was obtained with the cells harboring pSSRAherA. The establishment of two efficient selectable markers (pyrEF and hmg) was subsequently exploited for genetic analysis. A herA merodiploid strain of S. islandicus was constructed using pyrEF marker and used as the host to obtain pSSRNherA transformant with simvastatin selection. While the gene knockout (¿herA) cells generated from the herA merodiploid cells failed to form colonies in the presence of 5-fluoroorotic acid (5-FOA), the mutant cells could be rescued by expression of the gene from a plasmid (pSSRNherA), because their transformants formed colonies on a solid medium containing 5-FOA and simvastatin. This demonstrates that HerA is essential for cell viability of S. islandicus. To our knowledge, this is the first application of an antibiotic selectable marker in genetic study for a hyperthermophilic acidophile and in the crenarchaeal lineage.