The antiviral activity of antisense oligodeoxynucleotide phosphorothioates complementary to the tat gene, the gag mRNA, and the rev mRNA were studied in a long-term infection model. Three antisense oligonucleotides directed to the splice-acceptor site of the tat gene failed to suppress human immunodeficiency virus type 1 replication at 1 microM concentration in long-term culture. In contrast, two oligodeoxynucleotide phosphorothioates (28-mer) complementary to the gag and the rev mRNAs inhibited viral replication for > 80 days, and the antiviral activity was sequence- and length-dependent. In addition, after pretreatment of cells we could reduce the concentration of the antisense oligodeoxynucleotides by > 10-fold and still maintain the inhibition of viral replication. These results suggest that chemotherapy for human immunodeficiency virus type 1 infection with antisense oligodeoxynucleotide phosphorothioates may be achieved by an initial high-dose treatment followed by a lower maintenance dose.