We had previously reported that tetramethyl-O-NGDA (M4N), a synthetic derivative of the naturally occurring nordihydroguaiaretic acid (NDGA), is able to inhibit HIV Tat transactivation by blocking host Sp1 protein at the Sp1 cognate binding site on the HIV LTR promoter. The present studies were undertaken to examine whether M4N is able to inhibit the replication of herpes simplex virus (HSV), another Sp1-regulated virus. The results showed that in Vero cells, M4N inhibits at micromolar levels (IC50 = 43.5 microM) the expression of the herpes immediate early gene (alpha-ICP4), which is essential for HSV replication. An electrophoretic mobility shift assay, examining Sp1 binding to the alpha-ICP4 promoter, showed a significant inhibition of the control bands: 88% inhibition of the fast moving band (FMB) and 45% of the slow moving band (SMB), at 100 microM of drug concentration. Comparative studies between M4N and acycloguanosine (acyclovir, ACV) in cultured Vero cells revealed an interesting pattern in the drug sensitivity (IC50) and cytotoxicity (TC50) parameters. For M4N, the IC50 varied between 11.7 and 4 microM in 10 passages of HSV-1 and 4 passages of HSV-2 with no indication for a requirement of higher drug concentration. In contrast, for acyclovir, the IC50 increased from 7 microM in the first passage to 444 microM in the tenth passage of HSV-1, and >88 microM for the fourth passage of HSV-2, indicating a rapid build-up of drug resistance against acyclovir. While the selective index (SI), defined as the ratio: TC50/IC50, remained relatively constant for M4N; it dropped 60-fold for acyclovir in the endpoints of viral passages. Drug sensitivity for M4N toward the acyclovir-sensitive strain (sm44) and the acyclovir-resistant strain (ACV-10) of HSV-1 was similar, indicating no cross-resistance between M4N and acyclovir in their anti-HSV effects. These results may have an important clinical relevance since HSV has been shown to be a factor for spreading of HIV.