Neutrophils dominate acute inflammatory responses that generally evolve into chronic inflammatory reactions mediated by monocyte/macrophages and lymphocytes. The latter cell types also serve as major targets for human immunodeficiency virus type 1 (HIV-1). In this study we have investigated the role of neutrophil products, particularly cathepsin G, in HIV infection. Cathepsin G induced chemotaxis and production of proinflammatory cytokines by macrophages but not CD4⁺ T cells. Pretreatment with cathepsin G markedly increased susceptibility of macrophages but not CD4⁺ T cells to acute HIV-1 infection. When macrophages were exposed to pertussis toxin prior to cathepsin G treatment, the cathepsin G-mediated effect was almost abrogated, suggesting that enhancement of HIV-1 replication by cathepsin G requires Gi protein-mediated signal transduction. Although prolonged exposure to cathepsin G suppressed HIV infection of macrophages, serine protease inhibitors, which are exuded from the bloodstream later during inflammatory processes, neutralized the inhibitory effect. Neutrophil extracts or supernatants from neutrophil cultures, which contain cathepsin G, had effects similar to purified cathepsin G. Thus, cathepsin G, and possibly other neutrophil-derived serine proteases, may have multiple activities in HIV-1 infection of macrophages, including chemoattraction of monocyte/macrophages (HIV-1 targets) to inflamed tissue, activation of target cells, and increase in their susceptibility to acute HIV-1 infection.