To understand how fruit development is controlled, we have begun experiments to identify DNA sequences and proteins that regulate gene expression during tomato (Lycopersicon esculentum) fruit ripening. We have focused on the E8 gene because its transcription is responsive to ethylene hormone and is activated at the onset of fruit ripening. We report here that sequences required for ethylene-responsive and developmentally regulated E8 gene expression in transgenic tomato plants are contained on a 4.4 kb restriction fragment which includes sequences 2 kb 5' and 0.5 kb 3' to the gene. In addition, we have identified a DNA-binding factor that specifically interacts with DNA sequences that flank the E8 gene. This DNA-binding activity is low in unripe fruit and increases during fruit ripening. This factor also binds to the 5'-flanking region of another ethylene-responsive gene which is coordinately expressed during tomato fruit ripening. These data suggest that the DNA binding-factor may be involved in the regulation of gene expression during fruit ripening.