Previously, Lrp-like transcriptional regulator LysM from the hyperthermoacidophilic crenarchaeonSulfolobus solfataricuswas proposed to have a single target, thelysWXJKoperon of lysine biosynthesis, and a single effector molecule, l-lysine. Here we identify ∼70 novel binding sites for LysM in theS. solfataricusgenome with a LysM-specific nanobody-based chromatin immunoprecipitation assay coupled to microarray hybridization (ChIP-chip) andin silicotarget site prediction using an energy-based position weight matrix, and validate these findings within vitrobinding. LysM binds to intergenic and coding regions, including promoters of various amino acid biosynthesis and transport genes. We confirm that l-lysine is the most potent effector molecule that reduces, but does not completely abolish, LysM binding, and show that several other amino acids and derivatives, including d-lysine, l-arginine, l-homoarginine, l-glutamine and l-methionine and branched-chain amino acids l-leucine, l-isoleucine and l-valine, significantly affect DNA-binding properties of LysM. Therefore, it appears from this study that LysM is a much more versatile regulator than previously thought, and that it uses a variety of amino acids to sense nutritional quality of the environment and to modulate expression of the metabolic machinery ofSulfolobusaccordingly.