It has been suggested that expression of the genes encoding the alpha 4/beta 1 integrin increases during wound healing of the cornea. As a first step in understanding the mechanisms required to stimulate alpha 4 gene expression during this process, we defined the minimal upstream sequence required to direct basal promoter activity for this gene. Using deletion analyses of the alpha 4 gene upstream sequence, we identified two functionally important negative regulatory elements. Dimethylsulfate (DMS) methylation interference assays provided evidence for the binding of a single nuclear protein to tandemly repeated homologous cis-acting elements (designated alpha 4.1 and alpha 4.2) from the alpha 4 basal promoter that share the core sequence 5'-GTGGGT-3'. The formation of a protection only at alpha 4.1 in DNase I footprinting suggested that it is the primary target element for the binding of nuclear proteins. Three distinct nuclear proteins bound a double-stranded oligonucleotide bearing the DNA sequence of alpha 4.1 to produce specific DNA-protein complexes (R1 to R3) in gel-shift assays, from which that producing R3 was identified as the protein yielding DNase I protection at alpha 4.1. Detailed mutational analysis of alpha 4.1 and alpha 4.2 indicated that both elements positively regulate gene expression in primary cultures of corneal epithelial cells and Jurkat tissue culture cells, which is consistent with the deletion analysis. However, when transiently transfected into pituitary GH4C1, the alpha 4.2 mutants yielded increased chloramphenicol acetyl transferase activity therefore demonstrating that these elements have the ability to function either as positive or negative regulators of gene transcription in a manner that is dependent on the type of cell transfected.