The CD28 antigen has been recently demonstrated to be a costimulatory molecule and is expressed by almost all thymic and peripheral T cell receptor (TcR) alpha beta+ and gamma delta+ cells in the mouse system. We show here that expression of CD28 is heterogeneous among murine intestinal intraepithelial lymphocytes (IEL). Whereas some TcR alpha beta-expressing IEL subsets such as CD4+8- and CD4-8 alpha+ beta+ cells express CD28 at the same levels as their phenotypic counterparts in lymph node, other subsets of TcR alpha beta cells (including CD4-8 alpha+ beta- and CD4+8 alpha+ beta- cells) as well as TcR gamma delta+ IEL fail to express CD28. Parallel experiments using aged BALB/c-nu/nu mice indicated that CD28 expression patterns among IEL are quite similar to those of normal BALB/c mice. Furthermore, forward light scatter analysis showed that CD28- cells are considerably larger than CD28+ cells in the gut, although cycling cells were rare in both subsets. Finally CD28- cells in the gut did not proliferate or produce IL-2 upon stimulation by anti-CD3 monoclonal antibodies (mAb) and phorbol 12-myristate 13-acetate, whereas CD28+ cells in the gut and lymph nodes responded to these stimuli. The response of the CD28+ cells was enhanced by anti-CD28 mAb. These results suggest that CD28- IEL (CD4- 8 alpha+ beta- cells, and some CD4+ 8 alpha+ beta- cells) may follow a different developmental pathway from that of CD28+ IEL in a thymus-independent environment, and that expression of CD28 correlates with responsiveness of the cells to triggering via the TcR-CD3 complex.