A mAb directed against the CD3 molecule was used to identify a subset of CD3+, CD4-, CD8- T cells previously undefined in the peripheral lymphoid organs of the mouse. Biochemical analysis of CFD3+, CD4-, CD8- splenocytes revealed that the vast majority of these cells express one of at least two distinct CD3-associated TCR γδ heterodimeric structures, but no detectable TCRαβ. One disulfide-linked heterodimer (77 kDa) is composed of two chains of 45 to 46 and 32 kDa. The latter chain was immunoprecipitated with an anti-TCR C(γ)1/C(γ)2 antiserum and was not glycosylated. An antiserum produced against a peptide corresponding to the C-terminal region of the predicted C(γ)4 gene product immunoprecipitated additional heterodimers (80 to 90 kDa). One heterodimer, composed of disulfide-linked 41- to 45-kDa proteins (including a V(γ)/C(γ)4 component), is expressed on a T cell hybridoma, DN-1.21, which was derived from fused splenic CD3+, CD4-, CD8- T cells. Another V(γ)/C(γ)4-containing heterodimer is composed of disulfide-linked 46- to 47-kDa glycoproteins. These findings demonstrate that CD3+, CD4-, CD8- T cells present in the peripheral lymphoid organs express a variety of paired TCRγδ proteins. Unlike CD3+, CD4-, CD8- thymocytes, these cells express high levels of C(γ)4, but little, if any TCRαβ.