The antigen receptor of the T lymphocytes is one of the most complex eukaryotic membrane structures studied to date. The T cell receptor (TcR) consists of two disulfide‐linked glycoprotein chains (α/βor γ/δ) and is noncovalently associated with a group of small and invariable CD 3 proteins. Four CD 3 chains have been recognized: two highly homologous glycoproteins CD 3 γ and δ, the more distantly related nonglycosylated CD 3 E chain, and the nonglycosylated CD 3 ζ, the latter being present as a homodimer. The unraveling of the architecture of the TcWCD 3 complex is crucial to our understanding of the processes underlying its assembly, recognition and trans‐membrane signaling. The transmembrane orientation of the TcR chains and of CD 3 γ and CD 3 δ can be directly inferred from their primary structure, based on the presence of concensus N‐linked glycosylation sites N‐terminal of their transmembrane domains. This prediction can not be made, however, for nonglycosylated molecules like the CD 3 E chain. In order to determine the transmembrane orientation of CD 3 E, anti‐peptide antisera directed against the N‐termini of the human and murine CD 3 E chains were generated in rabbits. Both antisera stained intact T cells, demonstrating that the N‐terminus of the CD 3 E chain was located at the outer surface of the plasma membrane. The anti‐human CD 3 E peptide antiserum was found to be mitogenic for peripheral blood T cells, a finding previously reported only for monoclonal anti‐TcW CD 3 reagents. Using a novel transient expression system in murine T lymphocytes, the human CD 3 E chain could be expressed on the surface of CD 3+, but not CD 3−murine T cells, as indicated by fluorescence staining with the anti‐peptide antiserum. This experiment confirmed the specificity of the anti‐peptide antiserum and, perhaps more importantly, indicated that the human CD 3 E chain was correctly assembled in the murine CD 3 complex. Moreover, the anti‐human CD 3 monoclonal antibody UCHT1 was found to stain T cells expressing the human CD 3 E chain.