The present study was undertaken to characterize structure-function relationships of the rat galectin-3 gene promoter especially focusing on the promoter binding proteins included in livers injured with CCl4. Transcription start site determination identified a 66-nucleotide-long exon 1 of this gene. Transient expression analysis using a reporter luciferase gene assigned a region between -161 and -15 to the proximal promoter within the 1-kb region flanking the 5'-end of exon 1. The rat galectin-3 gene promoter possesses a Runx2 binding site and inverted repeats of Sp1 binding motifs in separate regions downstream from -117 as structures resembling those of the mouse galectin-3 gene promoter. The -161/-118 region bound two different proteins. One is a novel protein, a rat version of Purbeta that binds to a guanine nucleotide pair at -145 and -144 to modulate constitutive galectin-3 gene transcription. Southwestern blot analysis using the -161/-118 ligand revealed a signal of a 50-kDa protein in liver nuclear extracts from rats 48-h post-treatment with CCl4, but not in those from Ac2F cells and normal rat livers. The inducible nature of this protein suggested its distinctive role in galectin-3 induction in a liver injured with CCl4. E-box and peroxisome proliferator response element-like motifs reside on separate DNA strands from -140 to -135. Contribution of this segment to the regulation of galectin-3 gene transcription under pathological conditions was suggested, since a DNA ligand with the two motifs simultaneously mutagenized at -136 and -137 was not bound by the 50-kDa protein.