The membrane permeation of ganciclovir (DHPG)--a structural analogue of acyclovir (ACV) with activity against cytomegalovirus--was investigated in human erythrocytes at 37 degrees with an "inhibitor-stop" assay. DHPG influx was nonconcentrative, occurred without permeant metabolism, and was rate-saturable. While substantial inhibition of the influx of 13 microM DHPG occurred only in the presence of permeants of the purine nucleobase carrier, nucleosides and inhibitors of nucleoside transport markedly inhibited DHPG influx at higher DHPG concentrations (greater than or equal to 200 microM). Adenine and dilazep (a potent inhibitor of the nucleoside carrier) each inhibited the influx of DHPG only partially; when present together, however, they inhibited DHPG permeation completely. DHPG permeation via the purine nucleobase carrier (Km = 0.89 mM) was characterized by assessing influx in the presence of 1.0 microM dilazep. Adenine and ACV were shown to competitively inhibit this process, while DHPG (Ki = 0.90 mM) was found to competitively inhibit adenine influx. DHPG influx via the nucleoside carrier (Km = 14 mM) was characterized by assessing influx in the presence of 2 mM adenine. DHPG (Ki = 10 mM) also appeared to competitively inhibit the influx of 5-iodo-2'-deoxyuridine. These results indicate that DHPG permeates the human erythrocyte membrane primarily by the purine nucleobase carrier and secondarily by the nucleoside transporter.