A distinctive characteristic of human intestinal lymphocytes is their low responsiveness to stimuli of the CD3 pathway in vitro. This may be due to anergy resulting from CD3 stimulation without costimulation. Costimulatory molecules, such as HML-1, VLA-4, CD44, and CD28, may be lacking or unresponsive to ligation. Expression of these costimulatory markers was examined by immunofluorescent staining and flow cytometry. Their ability to costimulate intestinal lymphocytes was tested by measuring changes in proliferation, IL-2 production, and calcium ion mobilization. HML-1 was found on 87 +/- 8% of intraepithelial lymphocytes but only 52 +/- 10% of lamina propria lymphocytes; the density of expression was three times higher on the former. VLA-4 and CD44 were expressed on more than 80% of both lymphocyte types. CD28 was found on 31 +/- 26% of intraepithelial lymphocytes and 55 +/- 15% of lamina propria lymphocytes; virtually all CD4+ T cells and 15 to 26% of CD8+ T cells were CD28+. Of the four costimulatory molecules, ligation of only CD28 increased proliferation and IL-2 production; none affected calcium ion mobilization with stimulation through the CD3 pathway. The marker B7/BB1, a binding partner to CD28, was not present on lymphocytes or epithelial cells by flow cytometry. CD28 is the only one of these costimulatory molecules that enhanced CD3-induced functions of both intestinal lymphocyte types, but little ligand is available in vivo. Without coligation, CD3 activation may lead to anergy.