Peripheral blood T lymphocytes (PBT) proliferate more to anti-CD3 stimulation than to anti-CD2 stimulation. On the other hand, fresh, but not cultivated, intestinal intraepithelial lymphocytes (iIEL) exhibit a lower response to CD3 stimulation in comparison to CD2. The goal of this study was to show that the anti-CD3 T-cell response depends on the microenvironment and is independent of the origin of the lymphocytes. Cultured T-cell lines were stimulated with either an anti-CD3 mAb or an anti-CD2 mAb. Either conditioned supernatant from intestinal epithelial cell (IEC) lines or non- conditioned medium (negative control) was added. After 2 days cytokine production and proliferation were measured. Conditioned supernatant decreased the proliferative response of small and large bowel iIEL compared to controls (P = 0.04). In the same experiments, the cytokine production was non-significantly decreased. Immortalized iIEL, that are not regularly stimulated by their CD3 pathway, showed a similar decrease in proliferation (P < 0.001) and cytokine production (P = 0.01) when incubated with conditioned supernatant. Similar results were also obtained with a non-immortalized and an immortalized PBT line (P < 0.001). In a small bowel iIEL cell line, that exhibited a significant response to anti-CD2 stimulation, the proliferative response to anti-CD2 stimulation was preserved. Active conditioned supernatant could be generated from three independent IEC lines and a liver derived epithelial cell line, but not from a non-epithelial control cell line or two extraintestinal epithelial cell lines. We conclude that supernatants of cultured IEC contain soluble factor(s) that cause cultured iIEL and extraintestinal lymphocytes to behave like fresh iIEL. These results, therefore, support and extend the studies of others which suggest that the intestinal microenvironment mucosalizes lymphocytes.