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Naturally occurring protoberberine alkaloids, berberine and berberrubine along with 9-substituted derivatives of berberine were assessed for the anti-human immunodeficiency virus (HIV) activity. Berberine was found to be the most active compound with an EC₅₀ of 0.13 μM against HIV-1 NL4.3 virus in CEM-GFP cell lines. Berberrubine and two other compounds were found to be less active than berberine, at the same time they were less toxic than berberine. Enzyme based assay suggested that the anti-HIV activity of berberine and its analogs might be due to RTase inhibitory activity and some additional mechanisms.