The effects of a soluble trimeric CD40 ligand (CD40L) agonist on the expression of CD4 and CCR5 and on human immunodeficiency virus (HIV) type 1 entry into and replication in human macrophages were investigated. CD40L increased the number of CD4 and CCR5 antibody-binding sites and the percentage of CD4- and CCR5-expressing cells. Infection of CD40L-stimulated macrophages with HIV-1 resulted in a marked increase of viral DNA with respect to controls, as demonstrated by polymerase chain reaction assay. HIV-1 p24 antigen analysis showed that peak viral production did not differ between CD40L-stimulated macrophages and controls. However, because of a prolonged life span, overall viral output was increased in CD40L-stimulated cultures. In addition, CD40L down-regulated the antiviral efficacy of compounds that inhibit HIV-1 reverse transcriptase. In conclusion, CD40L stimulation of macrophages can contribute to plasma virus load and favor the establishment of a pool of latently infected macrophages that can be reactivated to release virus.