A thermophilic lipase (lipGRD) from Geobacillus sp. RD-2, isolated from a hot spring in Yunnan, China, was cloned and over-expressed in Escherichia coli. The function of the conserved residue, Tyr224, near the presumed temperature switch site was analyzed by site-directed saturation mutagenesis. The activity of the wild type lipGRD was optimal at 55A degrees C and pH 7.5, but that from mutant Y224C was optimally active at 35A degrees C, whereas Y224P lipase was optimally active at 65A degrees C. Furthermore, the latter lipase retained 60% of its activity after incubation at 65A degrees C for 5 h. The conserved residue Tyr224, which is close to the lid helix, is the key amino acid residue determining the thermostability of the thermostable lipase.