Intestinal intraepithelial lymphocytes (IEL) from mice are >80% CD3+ T cells and could be separated into four subsets according to expression of CD4 and CD8. In our studies designed to assess the functions of IEL, namely, cytokine production, it was important to initially characterize the various subsets of T cells that reside in IEL. The major subset was CD4-, CD8+ (75% of CD3+ T cells), which contained approximately 45 to 65% γ/δ TCR+ and 35 to 45% α/β TCR+ T cells. Approximately 7.5% of IEL T cells were CD4-, CD8- (double negative) and γ/δ+ population. On the other hand, CD4+, CD8+ (double positive) and CD4+, CD8- fractions represented 10% and 7.5% of CD3+ T cells, respectively, which were all α/β TCR+. Inasmuch as CD3+, CD4-, CD8+ T cells are a major subset of IEL which contain both γ/δ TCR or α/β TCR-bearing cells, the present study was focused on the capability of this subset of IEL T cells to produce the cytokines IFN-γ and IL-5. Both γ/δ TCR+ and α/β TCR+ IEL spontaneously produced IFN-γ and IL-5, although higher frequencies of cytokine spot-forming cells were associated with the α/β TCR+ subset. Approximately 30% of CD8+, γ/δ TCR+ cells produced both cytokines, whereas ~90% of α/β TCR+ T cells produced either IFN-γ or IL-5. Both γ/δ TCR+ and α/β TCR+ IEL possessed large quantities of cytokine-specific mRNA, clearly showing that these IEL were programmed for cytokine production. When IEL were activated with anti-γ/δ or anti-CD8 antibodies, higher numbers of IFN-γ and IL-5 spot-forming cells were noted. The present study has provided direct evidence that a major function of IEL involves cytokine production, and this is the first evidence that γ/δ TCR+ cells in IEL possess the capability of producing both IL-5 and IFN-γ.