We have used DNase I footprinting to characterize nuclear factors that bind to the light-responsive promoter of pea rbcS-3A, one member of the gene family encoding the small subunit of ribulose-1,5-bisphosphate carboxylase. A sequence-specific binding activity, designated 3AF1, binds to an AT-rich sequence present at the -45 region of the rbcS-3A promoter. A tetramer of the 3AF1 binding site, designated as Box VI, can form multiple complexes with tobacco leaf and root nuclear extracts. Mutations of 3 base pairs in Box VI severely reduce DNA-protein complex formation in vitro. The wild-type Box VI tetramer, but not the mutant tetramer, is active in transgenic tobacco plants when placed upstream of the cauliflower mosaic virus 35S promoter truncated at -90. These results correlate binding of 3AF1 to the in vivo function of Box VI. The Box VI tetramer/35S chimeric construct confers expression in diverse cell types and organs and its activity is not dependent on light. By using the Box VI tetramer as a probe to screen a cDNA expression library, we have obtained a putative cDNA clone for the 3AF1 DNA-binding activity. Lysogen extracts of Escherichia coli expressing the cDNA clone give sequence-specific complexes with Box VI. The deduced amino acid sequence of the protein encoded by the cDNA contains two stretches of about 100 residues that are 80% homologous. Moreover, in each of the two repeats, there is an arrangement of histidines and cysteines, which may be related to the two known types of zinc-finger motifs found in many DNA-binding proteins. Consistent with the expectation that metal coordination plays an important role in DNA binding by this protein, we found that 1,10-phenanthroline can abolish the formation of DNA-protein complexes. Interestingly, we found that the same treatment did not abolish the DNA binding activity of 3AF1 in crude nuclear extracts of tobacco. These data indicate that the nuclear 3AF1 activity is likely due to multiple DNA-binding proteins all interacting with Box VI in vitro. RNA gel blot analysis shows that multiple transcripts homologous to this cDNA clone are expressed in different tobacco organs.