The cellular source of interleukin-6 (IL-6) during infection of mice with Listeria monocytogenes was investigated both in vitro and in vivo. Peritoneal cells taken at intervals from infected mice and cultured in vitro without added stimulus produced high titers of IL-6 peaking 2 days postinfection in a time course similar to that observed in vivo. Adherent cells with the morphology of macrophages were a major source of this IL-6. Spleen cells similarly harvested at intervals and cultured with heat-killed Listeria or heat-killed Brucella organisms as specific and nonspecific stimuli, respectively, showed two distinct IL-6 responses: (i) an early-phase response up to 5 days after infection when IL-6 production was elicited by either a specific or nonspecific stimulus, and when depletion of T cells had no effect, and (ii) a later response 7 to 10 days after infection when very high levels of IL-6 were produced in response to a specific stimulus. This response was lost when T cells were depleted in vitro or in vivo or in spleen cell cultures from mice with severe combined immunodeficiency. However, studies in vivo failed to show an important role for T cells governing serum IL-6. We conclude that most of IL-6 detected in vivo is produced by nonlymphocytes. Whether IL-6 produced by T lymphocytes in local foci of infection has any role in resolution of that infection is unknown.