T cell-specific expression of human and mouse CD3delta is known to be governed by an enhancer element immediately downstream from the gene. Here we demonstrate by transgenic and in vitro studies that the murine CD3delta (mCD3delta) promoter prefers to be expressed in cells of the T lineage. Deletion analyses of a promoter segment (-401/+48 bp) followed by transient transfections indicate that upstream elements between -149 and -112 bp contribute to full expression of the gene. Furthermore, a core promoter region -37/+29 appears to contribute to a T cell specificity. Using substitution mutant scanning, four positive and one negative regulatory elements were found within the mCD3delta core promoter. The first two positive elements comprise two classical initiator-like sites, which recruit TFII-I, whereas a third contains a functional Ets binding site. Immediately adjacent to the observed transcription start site is a negative element that utilizes the transcription regulator YY1. The last positive regulatory element contains a potentially functional CREB binding site and the minor transcriptional start site. Because NERF-2, Elf-1, and Ets-1 are expressed preferentially in lymphocytes and because, in addition, YY1 represses the promoter activity strongly in non-T cells, we conclude that the combination of these transcription factors contributes to the T cell-specific expression pattern of mouse CD3delta.