The cis-acting elements located within 15 kb 5' of the murine CD4 gene transcriptional start site and the first intron of the CD4 gene have been investigated using deletion constructs. Our transient transfection data indicate that the expression of the murine CD4 gene is controlled by multiple positive and negative regulatory cis-acting elements. There are at least two cis-acting elements that have a positive effect on the expression of the CD4 gene and at least four regions of DNA that have a negative effect. The positive control elements are located about 13.5 kb 5' of the promoter and within the flanking sequences of the first intron. The DNA between the 5' enhancer and the promoter contains at least two regions that exert a negative effect on CD4 expression. In addition to the positive effect that the first intron has on CD4 expression, there are two regions within the first intron that have a negative effect. These two negative regulatory elements correspond to two T-cell-specific DNase I hypersensitive sites found in the first intron.