We examined the inhibitory activities of 10 polyether antibiotics on human immunodeficiency virus (HIV) type 1. These compounds caused concentration-dependent inhibition of HIV replication in primary infected cultures of human T-lymphoblastoid H9 cells. The ratio of 50% effective concentrations for cellular cytotoxicity (MTT assay) to antiviral activity (reverse transcriptase assay) was over 5. Anti-HIV activity was also observed in cultures of monocytic lineage U937 cells chronically infected with HIV.