DNase I footprinting assays were used to map sites of DNA-protein interaction in the promoter regions of three of the five genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase (rbcS) in tomato. Organ-specific differences in DNase I protection patterns were observed using nuclear extracts derived from cotyledons, leaves, young fruit, mature fruit, and roots of tomato, implying that organ-specific transcription of these genes is controlled at the level of DNA-protein interaction. The three genes, designated rbcS1, -2 and -3A are similarly expressed in cotyledons of dark-grown seedlings, in immature tomato fruit, and in leaves under conditions of water stress. These three genes share at least three DNA sequence motifs, including the G-box sequence, which are apparently not present in the other two tomato rbcS genes. We find protection of one or more of these sequences in the aforementioned organs, indicating that the corresponding DNA-binding proteins could function in directing differential expression of the genes, although functional studies would be required to establish this point. While most of the DNase I protections encompass previously identified conserved sequence motifs and their flanking sequence, we also observe protection of additional sequences, many of which occur in the region of the transcription start site.