Using a flow cytometric assay, we investigated neutrophil–Mycobacterium tuberculosis opsonophagocytosis and the impact of HIV-1–infected serum on this process. The mean (±SD) percentage of neutrophils internalizing bacilli after 30 minutes incubation was significantly reduced by pretreatment with anti-CD16 (18.2% ± 8.1%, P < 0.001) or anti-CD35 antibody (23.2% ± 10.6%, P < 0.05) versus anti-CD4 controls (29.9% ± 8.1%). Blocking CD88 or CD11a did not affect internalization. Using heat-inactivated serum, maximal internalization was lower using HIV-1–infected serum versus HIV-1–uninfected. Using non–heat-inactivated serum, internalization decreased more rapidly with sequential dilutions of HIV-1–infected versus HIV-1–uninfected serum. CD16 and CD35 are important for neutrophil internalization of M. tuberculosis, whereas HIV-1 infection adversely affects opsonophagocytosis.