The expression of the germ-line gene V gamma 1.1-C gamma 4 of the T cell receptor (TcR) gamma chain depends on interleukin (IL)-3 induction in hematopoietic cells, while in T cells, the rearranged gene is expressed constitutively. To understand the mechanism that controls TcR gamma gene expression, we cloned and characterized the structure and function of the V gamma 1.1-C gamma 4 TcR promoter. IL-3-dependent cell lines and T cell lines utilized the same transcriptional start sites. In chloramphenicol acetyltransferase (CAT) assays, the minimal 70-bp promoter confers strong transcriptional activity which is 50-60% of the Moloney long terminal repeat promoter activity. The 500-bp promoter region linked to the CAT gene exhibits IL-3 dependency similar to the endogenous TcR gamma gene. The immediate 3' and 5' flanking sequences inhibit the promoter activity two- to fourfold. The promoter lacks an obvious TATA box or CAAT box sequences, but contains a GC box in the untranslated region 3' to the promoter. The GC box is the core sequence of the element which binds Sp1-like proteins. Cloning of this Sp1 binding element in front of the thymidine kinase (TK) promoter and mutations generated in this site demonstrate its function as a silencer. Ultraviolet cross-linking analysis with the Sp1 binding site from the TcR gamma promoter revealed binding of a 90-100-kDa protein in a T cell line (EL-4) and 40-50 and 90-100-kDa proteins in FDC-P1 cells. The possible function of the Sp1-like protein in silencing the minimal promoter activity is discussed.