Fructose-1,6-bisphosphate aldolase from the thermophilic eubacteria, Thermus aquaticus YT-I, was cloned and sequenced. Nucleotide-sequence analysis revealed an open reading frame coding for a 33-kDa protein of 305 amino acids having amino acid sequence typical of thermophilic adaptation. Multiple sequence alignment classifies the enzyme as a class II B aldolase that shares similarity with aldolases from other extremophiles: Thermotoga maritima, Aquifex aeolicus, and Helicobacter pylori (49-54% identity, 76-81% homology). Tag FBP aldolase was overexpressed under tac promoter control in Escherichia coli and purified to homogeneity using heat treatment followed by two chromatographic steps. Yields of 40-50 mg of monodisperse protein were obtained per liter of culture. The quaternary structure is that of a homotetramer stabilized by an apparent al-amino-acid insertion sequence, The recombinant protein is thermostable for at least 45 min at 80 degreesC with little residual activity below 60 degreesC, Kinetic characterization at 70 degreesC, the optimal growth temperature for II aquaticus, indicates extreme negative subunit cooperativity (h = 0.32) with a limiting K-m of 305 muM. The maximal specific activity (V-max) is 46 U/mg at 70 degreesC.