T cells expressing T-cell receptor (TcR)-gamma delta and CD8 represent a significant population in mouse and chicken intra-epithelial lymphocytes (IEL) but represent a minor population in human IEL. We examined the TcR-gamma delta usage and co-expression of CD5, CD4, CD8 and major histocompatibility complex (MHC) class II on isolated sheep IEL and lamina propria lymphocytes (LPL), and compared them with the TcR-gamma delta + cells in peripheral blood, intestinal lymph and jejunal Peyer's patches (PP). There were a number of notable differences. TcR-gamma delta + cells comprised 18% of IEL and 10% of LPL. Among the population of TcR-gamma delta + IEL, 24% were CD8+ and 54% were CD5+, which contrasts with the TcR-gamma delta + cells in blood and intestinal lymph that were universally CD5+ CD4- CD8-. A notable feature of the IEL was the presence of distinct CD8+ and TcR-gamma delta + populations that lacked CD5. Also a high percentage of IEL and LPL were CD2+ and MHC class II+. Analysis of the expression of MHC class II on T-cell subsets, as an indicator of activation, showed that 60-95% of the various IEL and LPL subsets were MHC class II+ compared with only 5-40% in jejunal PP, lymph nodes, spleen and blood. Therefore, it is possible that the circulating TcR-gamma delta + and CD8+ cells that localize in the gut epithelium might become activated and stop the expression of CD5 under the influence of the local microenvironment. These cells appear not to emigrate while still expressing the TcR-gamma delta + (CD8+) CD5- MHC class II+ phenotype. Our data, together with those from other studies, show that there is much heterogeneity in the use of TcR-gamma delta and accessory T-cell molecules by IEL.