Using ferritin as a marker of reactive microglia, we demonstrated a close association between proliferation of reactive microglia and expression of human immunodeficiency virus type 1 (HIV-1) in brain tissue from autopsied cases of acquired immunodeficiency syndrome (AIDS). An increased number of ferritin-positive reactive microglia was observed in formalin-fixed paraffin-embedded brain sections from all 13 AIDS cases examined. Similar findings were observed in brain tissue from other neurological diseases (subacute sclerosing panencephalitis, herpes simplex encephalitis and multiple sclerosis). Multinucleated giant cells were found in 7 of the AIDS cases which were also intensely labeled for ferritin. Dual-label immunohistochemistry using anti-ferritin and cell-specific markers showed that ferritin-positive cells were distinct from astrocytes, neurons and endothelia using anti-glial fibrillary acidic protein (anti-GFAP), anti-neurofilament protein and Ulex europaeus agglutinin 1, respectively. In 5 AIDS brains, only ferritin-positive cells were shown to contain HIV-1 gp41 antigen using dual-label immunohistochemistry. In addition, HIV-1 RNA was localized in ferritin-positive reactive microglia but not in GFAP-positive astrocytes using immunohistochemistry combined with in situ hybridization. Ferritin-positive reactive microglia and multinucleated giant cells were co-labeled with the microglial marker, Ricinus communis agglutinin 1 (RCA-1). However, RCA-1 also extensively stained resting microglia only a few of which were co-labeled for ferritin. The density of ferritin-positive cells was correlated with the presence of HIV-1 RNA-positive cells in AIDS brain. Thus, ferritin immunoreactivity can be used as an activation marker of microglia in archival paraffin sections and reflects the extent of inflammation in HIV-1-infected brain.