Under normal circumstances most lymphoid cell populations do not exhibit strong proliferative reactions in culture unless provoked by antigen or mitogen. The autologous mixed lymphocyte reaction (AMLR) mediated by adult T cells is a relatively weak proliferative response that occurs in the absence of known heterologous stimuli. In this investigation we demonstrate that Peyer's patch (PP) cells possess an inherent capacity to commence dividing in vitro and to display an exceptionally vigorous AMLR. The magnitude and kinetics of this spontaneous proliferation resemble that of a secondary response to a strong mucosal immunogen such as reovirus type 1/Lang. Analysis of the cellular components of the PP cultures implicates CD4+CD8- T cells as the major responding population and dendritic cells (DC) as stimulators. Mixing experiments indicate that spleen contains a cell population which can stimulate PP T cells, albeit to a lesser extent than PP cells. Similarly, splenic T cells have a reduced but significant capacity to respond to PP DC, in comparison to PP T cells. These differences suggest the possibility that there may be a decreasing gradient of antigenicity between the gut and the spleen which is reflected in the spontaneous activity of PP versus splenic T cells in vitro. We propose that PP cells are in fact responding in vitro to heterologous antigens derived from food, enteric microbes and other environmental sources. This notion is supported by the observation that PP cells from antigen-minimized germ-free mice fail to proliferate spontaneously in culture.