Previous studies from this laboratory have suggested a role for cell-mediated immunity in host defense against Legionella pneumophila. In this paper, cell-mediated immunity to L. pneumophila in patients recovered from Legionnaires' disease was studied by examining patient mononuclear cell responses to L. pneumophila antigens. Patient mononuclear cells were assayed both for their capacity to respond to L. pneumophila antigens with the production of cytokines that activate monocytes, as measured by monocyte inhibition of L. pneumophila multiplication, and for their capacity to respond with proliferation, as measured by [(3)H]thymidine incorporation. Patient mononuclear cells incubated with formalin-killed L. pneumophila generated cytokines (supernatants) that were capable of activating in vitro freshly explanted monocytes from a person without historical or serological evidence of L. pneumophila infection (nonpatient). Such activated nonpatient monocytes inhibited the intracellular multiplication of L. pneumophila, and the degree of inhibition was proportional to the concentration of supernatant added. Patient mononuclear cells incubated with 5 x 10(6) - 5 x 10(8) formalin-killed L. pneumophila/ml for 4 d produced maximally potent supernatants; supernatants generated in flat-bottomed wells were equivalent in potency to supernatants generated in cone-shaped wells. Patient L. pneumophila-induced mononuclear cell supernatants were less potent than patient concanavalin A-induced mononuclear cell supernatants. Patient mononuclear cells also responded to formalin-killed L. pneumophila with proliferation (lymphoproliferation). Patient mononuclear cells responded more strongly to L. pneumophila antigens than mononuclear cells of age- and sex-matched nonpatients, as measured by both assays; responses to concanavalin A were comparable. Mononuclear cells from patients recovered from Legionnaires' disease responded more strongly to L. pneumophila than to Mycobacterium leprae antigens, whereas mononuclear cells from patients with tuberculoid leprosy responded more strongly to M. leprae antigens. These findings indicate that cell-mediated immunity to L. pneumophila develops in patients with Legionnaires' disease and, taken together with previously reported findings, that cell-mediated immunity plays a major role in host defense against L. pneumophila. The monocyte activation assay described in this paper has general applicability for the study of monocyte and mononuclear cell effector functions in selected patients. The assay may be used to study (a) the capacity of a patient's monocytes to be activated to an antimicrobial potential by a standard preparation of cytokines and (b) the capacity of a patient's mononuclear cells to generate such monocyte-activating cytokines in response to a mitogen or antigen.