The receptor for antigen on the surface of T lymphocytes consists of a variable disulfide-bridged hetero-dimer (TCR-alpha/beta or -gamma/delta) associated with invariant CD3 proteins (CD3-gamma, -delta, -epsilon, and -zeta). The genes coding for the CD3 proteins are expressed in the earliest recognizable thymocytes, preceding the rearrangement and expression of the TCR genes. The isolation, characterization, and in vitro expression of the murine CD3-epsilon gene, as reported here, represent obligatory steps toward our understanding of the complex rules that govern T-cell-specific gene expression. The CD3-epsilon gene was transcribed from a non-TATA promoter and consisted of eight exons, two of which were unusually small (18 and 15 base pairs). The transmembrane exon was found to be homologous to the transmembrane exons of the CD3-gamma and CD3-delta genes. In transient-transfection experiments, a genomic fragment comprising 4 kilobases of upstream sequence and extending into the second exon sufficient to drive the expression of a reporter gene in murine T cells.