An extremely thermostable UDP-GlcNAc pyrophosphorylase has been purified from Thermus caldophilus GK24 by chromatographic methods including ion-exchange, hydrophobic interaction, and affinity chromatographies. The specific activity of the enzyme was enriched 41.8-fold, with a recovery of 2%. The molecular mass of the enzyme was 41 kDa by SDS/PAGE and 45 kDa by gel-filtration chromatography. The activity was maximum at 86 degrees C and its half-life at 95 degrees C was 30 min. Its optimum pH was 6.9 in the presence of Mg2+ ions. A biochemical study showed that UDP-GlcNAc pyrophosphorylase activity could be enhanced by fructose 1-phosphate, a precursor of UDP-GlcNAc. The enzyme showed a broad substrate specificity with sugar 1-phosphates, including glucose 1-phosphate, GlcNAc-1-P and xylose 1-phosphate. The enzyme was therefore named UDP-sugar pyrophosphorylase. The N-terminal and internal peptide sequences were determined and compared with known sequences from various sources. It was found that N-terminal sequence is similar to that of UDP-GlcNAc and UDP-glucose pyrophosphorylases from other bacterial sources.