Mycobacterium avium is a common opportunistic pathogen in immunocompromised patients such as those infected with human immunodeficiency virus. Although M. avium is an intracellular organism replicating predominantly in macrophages, disseminated M. avium infection is seen in AIDS patients with CD4+ cell counts of <50 cells/μl, suggesting a possible involvement of a T cell-macrophage interaction for the elimination of M. avium. To determine whether CD40-CD40 ligand (CD40L) interactions play a role in M. avium infection, we studied the ability of CD40L to restrict M. avium replication in human monocyte-derived macrophages (MDM) in vitro. MDM were infected with M. avium and cocultured with CD40L-transfected 293 cells for 7 days. Intracellular growth of M. avium in these MDM was assessed by colony counting. CD40L-expressing cells inhibited growth of M. avium in MDM by 86.5% ± 4.2% compared to MDM cultured with control cells. These findings were verified by assays using purified, soluble recombinant human CD40L (CD40LT). CD40LT (5 μg/ml) inhibited intracellular growth of M. avium by 76.9% ± 18.0% compared to cells treated with medium alone. Inhibition by CD40LT was reduced by monoclonal antibodies (MAbs) against CD40 and CD40L. The inhibitory effect of CD40LT was not accompanied by enhancement of interleukin-12 (IL-12) production by M. avium-infected MDM, while CD40L- expressing cells stimulated IL-12 production by these cells. Treatment of M. avium-infected mice with MAb against marine CD40L resulted in recovery of larger numbers of organisms (0.8 to 1.0 log) from the spleens, livers, and lungs of these animals compared to infected mice which received normal immunoglobulin G. These results indicate that CD40-CD40L signaling may be an important step in host immune response against M. avium infection.