Sulfolobus solfataricus P2 was shown to survive on ethanol at various concentrations (0.08-3.97% w/v) as the sole carbon source. The highest ethanol consumption rate was 15.1 mg/L/hr (via GC-MS analysis) in cultures grown on 0.79% w/v ethanol. In vivo metabolic labeling, using 13C universally labeled ethanol, provided evidence for both ethanol uptake and metabolic utilization. Results obtained from isobaric mass tag-facilitated shotgun proteomics (iTRAQ) indicate that on average, 21 and 31% of the 284 proteins identified (with > or = 2 MS/MS) are increased and decreased expression in ethanol cultures compared to glucose control cultures. Preliminary analysis shows >2-fold increase of the zinc-dependent alcohol dehydrogenase, ADH-10 (SSO2536), and the putative ADH-2 (SSO0764) in both translational and transcriptional data (using quantitative RT-PCR), suggesting both proteins are integral to ethanol metabolism. Evidence that ethanol was catabolised into central metabolism via acetyl-CoA intermediates was further indicated by another >2-fold increase in protein expression levels of various acetyl-CoA synthetases. The decreased expression (>2-fold) of isocitrate dehydrogenase at the protein level suggests that the ethanol grown cultures shifted toward the glyoxylate cycle. Subsequently, the activity of ADH-2 was confirmed by overexpression in Escherichia coli, with the resultant purified in vitro enzyme exhibiting an activity that increased with temperature up to 95 degrees C, and giving a specific activity of 1.05 U/mg.