Studies of the mechanism of action of michellamine B, a novel anti-human immunodeficiency virus (HIV) alkaloid from the tropical plant Ancistrocladus korupensis, have revealed that the compound acts at two distinct stages of the HIV life cycle. The compound had no direct effect on HIV virions and did not block the initial binding of HIV to target cells. Postinfection time course studies revealed that the agent partially inhibited HIV-induced cell killing and syncytium formation when added up to 48 h following acute infection; however, vital reproduction was fully inhibited only when the compound was added immediately after infection. Time-limited treatments of HIV-infected cells revealed that michellamine B had to be present continuously to provide maximum antiviral protection. HIV replication in cells in which infection was already fully established or in chronically infected cells was unaffected by michellamine B. Biochemical studies showed that michellamine B inhibited the enzymatic activities of reverse transcriptases (RTs) from both HIV type 1 and HIV type 2 as well as two different nonnucleoside drug-resistant RTs with specific amino acid substitutions. In addition, human DNA polymerases α and β were inhibited by the alkaloid. Michellamine B exerted a potent dose-dependent inhibition of cell fusion in two independent cell-based fusion assays. Thus, michellamine B acts both at an early stage of the HIV life cycle by inhibiting RT as well as at later stages by inhibiting cellular fusion and syncytium formation.