Vertebrates possess three isozyme forms of fructose diphosphate aldolase. We have isolated two overlapping chicken genomic clones which encode the liver-specific form of this enzyme; we have identified the 5' and 3' ends of this gene by a combination of primer extension analysis and S1 mapping; and we have determined the entire nucleotide sequence of this gene including 1400 base pairs (bp) of sequence from the regions flanking the 5' and 3' ends of the gene. The transcriptional unit for the aldolase B gene spans 8700 bp and contains eight intervening sequences, including a 4600-bp intron in the 5' non-coding region. On the basis of results from Southern genomic hybridizations, the aldolase B gene appears to be present only once per haploid genome. No differences were detected in the mRNA structure between RNA from three tissues expressing aldolase B (liver, kidney, and small intestine). Various features of the 5' flanking region are discussed, including a partial homology with the 5' noncoding region from rabbit aldolase A.