Previously, we isolated the human AE2 (SLC4A2) gene, a member of the sodium-independent anion exchanger family. Rat ortholog of this gene was reported to drive alternative transcription yielding N-terminal variants of the AE2a message. We thus analyzed the human AE2 gene in this regard. Using HepG2 cells, two alternative first exons, each splicing to exon 3 in alternative transcripts, were found to be transcribed from overlapping sequences of intron 2. Exon 1b₁ corresponds to the rat variant “b” and encodes three initial residues (MTQ) in AE2b₁ isoform that replace the first 17 amino acids of AE2a protein, while the novel exon 1b₂ encodes eight initial residues (MDFLLRPQ) in AE2b₂ isoform. The relative abundance of AE2b₁ and AE2b₂ mRNAs was about 10% of AE2a mRNA each. Alternate promoter sequences have multiple potential binding motifs for liver-enriched factors, and dual-luciferase assays indicated that they possess the ability for driving transcription in transiently transfected HepG2 cells. Tissue survey showed that expression of human AE2b₁ and AE2b₂ transcripts is restricted to liver and kidney, while AE2a mRNA was encountered in all examined tissues. Our findings reveal a characteristic tissue-specific expression of two N-terminal variants of human AE2 from overlapping sequences within intron 2, one of which is a novel isoform.