The antiviral activity of mismatched dsRNA of the form poly(I):poly(C12-U)n (Ampligen) against the human immunodeficiency virus type 1 (HIV-1) was investigated by RNA-RNA and RNA-DNA hybridizations. Mismatched dsRNA delayed the appearance of newly transcribed HIV-1 RNA as detected by liquid dot-blot hybridization in cultures of H9 T-lymphoblastoid cells following virus challenge. The appearance of proviral DNA as detected by Southern hybridization following virus challenge in H9 cells was also delayed. Mismatched dsRNA had no effect in syncytium inhibition assays performed by fusing MT-2 cells with H9/HTLV-IIIB cells. These results suggest that the in vitro anti-HIV-1 activity of mismatched dsRNA occurs, at least in part, at an early stage in the viral replication cycle following initial gp120-CD4 binding.