Genes encoding helianthinin, the major seed protein in sunflower, are highly regulated. We have identified putative cis-acting and trans-acting elements that may function in the control of helianthinin expression. A 404-base pair DNA fragment on the sunflower helianthinin gene HaG3D, located 322 base pairs from the transcriptional start site, enhanced beta-glucuronidase expression in transgenic tobacco embryos. Sequences within this fragment were found to bind nuclear proteins present in both sunflower embryo and hypocotyl nuclear extracts. The binding site was localized by phenanthroline-copper ion footprinting experiments to A/T-rich sequences located from -705 to -654. Binding competition experiments revealed that these sunflower proteins also bind to upstream promoter sequences from another helianthinin gene (HaG3A) and two other plant embryo-specific genes, carrot DcG3 and French bean phaseolin. However, sequences of the cauliflower mosaic virus 35S promoter/enhancer complex failed to compete for its binding. Phenanthroline-copper ion footprinting experiments showed that the binding sites for the sunflower proteins in HaG3A (-1463 to -1514 and -702 to -653) and in phaseolin (-671 to -627) are also very A/T-rich, have similar sizes, and are located at similar distances from their respective promoters.