We have previously described conjugates of L-arginine with aminoglycosides (AAC) that have shown anti-human immunodeficiency virus type 1 (HIV-1) activity in in vitro cell culture systems. Here, we extend our report to a novel neomycin B-arginine conjugate (NeoR) that has shown up to 30-fold increased potency over previous AAC compounds. NeoR inhibited the replication of both R5 and X4 strains of HIV-1 in cells expressing the appropriate coreceptor or peripheral blood mononuclear cells. In lymphoid tissue ex vivo, NeoR blocked the replication of the dualtropic strain 89.6 suggesting anti-HIV activity of AAC on the site of in vivo virus replication. NeoR blocked the binding of HIV particles to lymphoid cells and was also able to antagonize the activity of the CXCR4 receptor so it may prevent the emergence of X4 HIV-1 strains. Nevertheless, in a cellular assay, we were unable to detect anti-Tat dependent transactivation activity as previously suggested for this family of compounds.