The synthesis, phosphorylation by guanylate kinase, anti HIV- 1 and anti-herpesvirus activity of two acyclic dienyl phosphonate derivatives of guanine are described. (E)-9-(5-phosphono-3-methylene-4 pentenyl)guanine (4) was identified as an excellent substrate of guanylate kinase and a significant inhibitor of HIV-1 replication. We report here the synthesis of 4 and of its shorter chain analogue 3, their substrate properties for guanylate kinase as well as their antiviral activity profile. Compound 4 proved to be an excellent substrate of guanylate kinase, being phosphorylated as efficiently as the natural substrate GMP. In MT-4 and C-8166 cells, compound 4 inhibited HIV-1 replication with IC50 values of 49 and 8 aM, respectively, and was more than 5 times less cytotoxic to MT-4 cells than PMEA. In contrast, its shorter chain analog 3 is a relatively poor substrate of guanylate kinase, being a 57-fold less efficient substrate than 4, did not inhibit HIV-1 replication at 100 BM in MT-4 cells, and showed some activity only against HSV-1. In conclusion, we have shown that acyclic dienyl phosphonate derivatives of guanine can act as substrates of guanylate kinase. Compound 4 was found to be an excellent substrate of guanylate kinase and a significant inhibitor of HIV replication. These examples illustrate the potential of the dienyl phosphonate moiety as a surrogate of the phosphate group. In addition, the antiviral activity of such compounds could be related, at least in part, to their ability to serve as substrates for guanylate kinase.