A series of 2'-substituted derivatives of 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG, 1) have been synthesized and evaluated in vitro for anti-human immunodeficiency virus (HIV) activity in the XTT assay and for anti-herpes activity in the plaque reduction assay. It has been observed that the anti-HIV activity of these derivatives depends on the size and the nature of the substituent as well as the chirality at the 2'-position of PMEG. In addition, these compounds generally demonstrated greater activity against HIV than herpes viruses. The most interesting analogues which emerged from these studies are (R)-2'-(azidomethyl)-PMEG [(R)-5] and (R)-2'-vinyl-PMEG [(R)-11]. The former showed anti-HIV activity with an IC50 of 5 microM and a cytotoxicity (CC50) greater than 1.4 mM in CEM cells. The latter has an IC50 of 13 microM for anti-HIV activity and a CC50 of greater than 1.6 mM. Furthermore, we have demonstrated that replacement of the guanine base of these 2'-substituted PMEG analogues with cytosine drastically reduces anti-HIV and anti-herpes activity.