Kijimicin represents an important type of ionophore compound. In veterinary medicine, it is becoming important as anticoccidiostatic agent and feed supplement. We examined Kijimicin for its HIV inhibitory activity. The compound exhibited concentration-dependent inhibition of HIV replication in primary infected cultures of human T-lymphoblastoid H9 cells. Substantial inhibition of viral replication was observed at concentrations of Kijimicin that showed little cytotoxicity. The ratio of IC50 values for the MTT to RT assays was 40. Anti HIV activity was also observed in cultures of monocytic lineage U937 cells chronically infected with HIV. Moreover, in attempting to define the inhibitory mechanism of Kijimicin, we investigated its effect on each step of HIV replication. The infectivity of progeny viral particles was reduced by Kijimicin treatment. This decrease may be due to incompletely glycosylated forms of gp120.