Murine intestinal intraepithelial lymphocytes (IEL) have been shown to contain subsets of α/β TCR+ and γ/δ TCR+ T cells that spontaneously produce cytokines such as IFN-γ and IL-5. We have now determined the nature and cell cycle stage of these cytokine-producing T lymphocytes in IEL by using IFN-γ- and IL-5-specific ELISPOT assay, cytokine-specific mRNA-cDNA dot-blot hybridization and polymerase chain reaction, and flow cytometry (FACS) for DNA analysis. When CD3+ T cells from IEL of normal C3H/HeN mice were separated into low and high density fractions by discontinuous Percoll gradients, IFN-γ and IL-5 spot-forming cells were only found in the former population. Analysis of mRNA for these cytokines by both IFN-γ- and IL-5- specific dot-blot hybridization and polymerase chain reaction revealed that higher levels of message for IFN-γ and IL-5 were also seen in the low density fraction. However, cell cycle analysis of these two fractions by FACS using propidium iodide showed a similar pattern of cell cycle stages in both low and high density populations (G0 + G1 ~96 to 98% and S/G2 + M ~2 to 4%). Finally, mRNA from γ/δ TCR+ and α/β TCR+ T cells in both low and high density fractions of IEL were analyzed for IFN-γ and IL-5 message by polymerase chain reaction. After 35 cycles of amplification, both γ/δ TCR+ and α/β TCR+ T cells in the low density fraction expressed higher levels of message for these two cytokines when compared with the high density population. These results have now shown that both γ/δ and α/β TCR+ IEL can be separated into low and high density subsets and both fractions possess a similar stage of cell cycle. However, only the low density cells (in G1 phase) of both γ/δ and α/β TCR types possess increased cytokine-specific mRNA and produce the cytokines IFN-γ and IL-5. Our results suggest that α/β TCR+ and γ/δ TCR+ IEL can produce cytokines without cell proliferation.