Intra-epithelial lymphocytes (IEL) in murine small intestine were analysed for the presence of cell-surface antigens and T-cell receptor allotype in normal and athymic BALB/c mice by immunoperoxidase histochemistry on frozen sections and immunofluorescence on isolated IEL. In frozen sections, IEL of normal mice were 97.7% CD45+, 93.5% CD3+, 46.2% Thy-1+, 91.1% CD8+, 10.7% CD4+ and 21.1% KJ16+ (V beta 8.1 and 8.2). FACS analysis of isolated IEL confirmed the level of KJ16 expression and also demonstrated that 25% of IEL were F23.1+ (V beta 8.1-8.3). Immunofluorescent double-staining revealed a skewed distribution of T-cell receptor (TcR) expression on Thy-1+ and Thy-1- IEL. KJ16 and F23.1 were expressed on 25.9% and 32.7% of Thy-1+ IEL, respectively; however, the frequency of V beta 8 expression was diminished on Thy-1- IEL (4.1% KJ16+ and 12.1% F23.1+). IEL are present in athymic mice, but at reduced levels. In frozen sections these cells were 91.9% CD45+, 69.5% CD3+, less than 1% Thy-1+, 83.6% CD8+, less than 1% CD4+ and less than 1% KJ16+. Thus it appears that in normal mice there may be two distinct lineages of IEL, a thymus-dependent Thy-1+ population which utilizes the alpha beta T-cell receptor and a thymus-independent Thy-1- population (represented in athymic mice), which may possibly utilize the alternative gamma delta TcR.