The sequence element of box II (GTGTGGTTAATATG) is a regulatory component of a light-responsive element present within the upstream region of pea rbcS-3A. The nuclear protein GT-1 was defined previously as a DNA binding activity that interacts with box II. Here, we describe the isolation and characterization of cDNA sequences that encode a DNA binding protein with specificity for this element. The recombinant protein, tobacco GT-1a, shows similar sequence requirements for DNA binding to nuclear GT-1, as assayed by its ability to interact with previously defined 2-bp scanning mutations of box II, and is shown to be immunologically related to nuclear GT-1. The predicted structure of the 43-kD protein derived from the cDNA sequence suggests the presence of a novel helix-helix-turn-helix (HHTH) motif. Comparison between the predicted protein sequence encoded by the tobacco GT-1a cDNA and that of another GT binding protein, rice GT-2, reveals strong amino acid conservation over the HHTH region; this motif appears to be involved in the interaction between the recombinant protein and box II. Genomic DNA gel blot analysis indicated the presence of a small gene family of related sequences within the tobacco nuclear genome. RNA gel blot analysis of tobacco mRNA using the isolated cDNA as a probe showed that transcripts are present in several tissues, including both light-grown and dark-adapted leaves.