Murine CD3+, CD4-, CD8-peripheral T cells, which express various forms of the TCR-γδ on their cell surface, have been characterized in terms of their cell-surface phenotype, proliferative and lytic potential, and lymphokine-producing capabilities.Three-color flow cytofluorometric analysis demonstrated that freshly isolated CD3+,CD4-,CD8- TCR-γδ lymph node cells were predominantly Thy-1+,CD5 dull,IL-2R-,HSA-,B220-, and approximately 70% Ly-6C+ and 70% Pgp-1+. After CD3+,CD4-,CD8- splenocytes were expanded for 7 days in vitro with anti-CD3-ε mAb (145-2C11) and IL-2, the majority of the TCR-γδ cells expressed B220 and IL-2R, and 10 to 20% were CD8+. In comparison to CD8+ TCR-αβ T cells, the population of CD8+ TCR-γδ-bearing T cells exhibited reduced levels of CD8, and about 70% of the CD8+ TCR-γδ cells did not express Lyt-3 on the cell surface. Functional studies demonstrated that splenic TCR-γδ cells proliferated when stimulated with mAb directed against CD3-ε, Thy-1, and Ly-6C, but not when incubated with an anti-TCR Vβ8 mAb, consistent with the lack of TCR-αβ expression. In addition, activated CD3+,CD4-,CD8- peripheral murine TCR-γδ cells were capable of lysing syngeneic Fcr-bearing targets in the presence of anti-CD3-ε mAb and the NK-sensitive cell line, YAC-1, in the absence of anti-CD3-ε mAb. Finally, activated CD3+,CD4-,CD8-,TCR-γδ+ splenocytes were also capable of producing IL-2, IL-3, IFN-γ, and TNF when stimulated in vitro with anti-CD3-ε mAb.