The tomato (Lycopersicon esculentum) gene family for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RBCS) has been investigated to determine the role of promoter regions and DNA-protein interactions in the differential organ-specific transcription of individual genes. Transgenic plants expressing RBCS-promoter-beta-glucuronidase fusion genes have confirmed that promoter fragments ranging from 0.6 to 3.0 kb of the RBCS1, RBCS2, and RBCS3A genes were sufficient to confer the temporal, organ-specific, and differential expression pattern observed for the endogenous genes. The individual temporal and organ-specific beta-glucuronidase enzyme activities closely reflect the qualitative and quantitative transcription activities of the respective RBCS genes, including the strongly reduced activity of RBCS3A (L.A. Wanner, W. Gruissem [1991] Plant Cell 3: 1289-1303). In particular, tissue-specific activity of all three promoters is similar in developing fruit, with high activity in the locular tissue and extremely reduced activity in the pericarp. This specific pattern of gene activity was further substantiated by in situ analysis of RBCS mRNA levels. Together, the data suggest an interesting correlation between RBCS gene activity and sink strength in different fruit tissues. DNA-protein interaction studies have revealed a novel fruit-specific DNA-binding protein called FBF that specifically interacts with a sequence element directly upstream of the G-box in the RBCS3A promoter. FBF binding thus correlates with the reduced activity of this promoter in developing tomato fruit, rendering it a candidate for a fruit-specific negative regulator of transcription in tomato.