Symmetric bis(naphthalenesulfonic acid) derivatives containing a variety of spacers have been synthesized and evaluated for anti-HIV-1 activity in four assay systems. In the assay that measured inhibition of HIV-1-induced cytopathogenicity using a laboratory strain (HTLV-IIIB), a hexamethylene and octamethylene spacer derivative of 4-amino-5-hydroxy-2,7-naphthalenedisulfonic acid emerged as the most potent derivatives. The hexamethylene spacer analog exhibited an in vitro therapeutic index that was > 120. Selected derivatives were tested in the giant cell formation assay. In this assay, the most potent derivative was, again, the hexamethylene compound. Evaluation of selected derivatives against a clinical isolate of HIV-1 (HE strain) revealed that the hexamethylene derivative was the most potent compound. In the assay that measured the inhibition of HIV-1-induced cytopathogenesis in human peripheral blood lymphocytes, the hexamethylene compound emerged as the most active derivative, demonstrating a 50% inhibitory concentration of 1.3 microM. These studies clearly demonstrate that certain naphthalenesulfonic acid moieties when coupled to specific spacers were synergistic in producing anti-HIV-1 activity at nontoxic concentrations. In the 4-amino-5-hydroxy-2,7-naphthalenedisulfonic acid series, shortening of the spacer length, preferably with a flexible polymethylene chain, was highly beneficial for increasing anti-HIV-1 potency.