Treatment of acutely infected human brain cell and enriched microglial cell cultures with diazepam inhibited human immunodeficiency virus type 1 (HIV-1) p24 antigen expression. Similarly, diazepam suppressed HIV-1 expression in chronically infected promonocytic (U1) cells and acutely infected monocyte-derived macrophages, and this antiviral activity was associated with decreased activation of nuclear factor kappa B.