To study the structure/function relationships of human aldolase isozymes, particularly isozyme-specific functions, we constructed Escherichia coli expression plasmids for six BA chimeric enzymes (BA34, BA108, BA137, BA212, BA306, and BA306*), each composed of the N-terminal side of isozyme B and the C-terminal side of isozyme A, and one BAB chimeric enzyme which contains a fragment of isozyme A (residues 213-306) inserted in between the N-terminal and the C-terminal fragments of isozyme B. They were transfected into E. coli, and the generated enzymes were characterized. This study reveals the following. (i) For isozyme A, the C-terminal Tyr-363 and the N-terminal region bearing isozyme group-specific sequences 1-3 and Lys-107 (the C-6 phosphate-binding site) are responsible for the higher catalytic activity toward fructose 1,6-bisphosphate, which is 7 times higher than that of aldolase B. Conversely, an internal region spanning positions 108-212 is required for the lower activity toward fructose 1-phosphate. (ii) For isozyme B, an internal sequence spanning positions 108-212 which includes some isozyme B-specific residues and a postulated C-1 phosphate-binding site (Lys-146 or Arg-148) is responsible for a higher catalytic activity toward fructose 1-phosphate, which is 8-10 times that of isozyme A. The more upstream sequence containing positions 1-107 is responsible for the lower catalytic activity toward fructose 1,6-bisphosphate. (iii) At least residues 212-306, composing a long stretch near the active-site Lys-229 and highly conserved among isozymes A, B, and C, may be required for the basal framework of the aldolase molecule to exhibit the activity common to the three isozymic forms.