In the mouse gene encoding the protein galectin-1, transcription initiation at the +1 site is directed by a TATA box. Here we show that a consensus Inr element (TCCAGTT), which spans residues -34 to -28 and overlaps the TATA box, directs RNA initiation also from a previously uncharacterized site located at position -31. Upstream transcripts are polyadenylated and contribute to more than half of the galectin-1 mRNA population in all tissues analyzed. The promoter architecture is evolutionarily conserved to man, and galectin-1 mRNA size variants accumulate also in human HeLa cells. The 5' end terminus of the transcripts initiated at residue -31 is extremely GC-rich, and may fold into a relative stable hairpin which could influence translation and thus modulate the intracellular levels of galectin-1. The interval -63/+45 contains sufficient information to ensure RNA initiation from both -31 and +1 sites, and a Sp1 site spanning residues -57 to -48 is crucial for promoter functioning. The unusual overlap of core promoter elements suggests that RNA initiation from the -31 and the +1 sites may take place in a sequential manner.