Antiviral therapy of primary and recurrent infections with human cytomegalovirus is reserved for severe manifestations and faces several limitations. Presently candidates for novel drugs with lower adverse side effects and a minimized frequency of resistance formation are under investigation. Here we demonstrate that artesunate, an antimalaria drug with highly valuable pharmacological properties, possesses antiviral activity. A concentration-dependent inhibition of the replication of human cytomegaloviruses with wild-type phenotype was demonstrated in several cell lines. Inhibition was quantified using recombinant green fluorescent protein expressing virus variants. The IC50 values were in the same range for ganciclovir-sensitive and ganciclovir-resistant human cytomegalovirus, as calculated with 5.8+/-0.4 microM and 6.9+/-0.2 microM, respectively. This indicated a strong antiviral potential and a lack of cross-resistance. The optimal antiviral concentrations of artesunate were separable from those inducing cytotoxicity. In addition, the replication of viruses from three genera was seen to be artesunate-sensitive to varying degrees. This suggests a mechanism linked to cellular activation pathways. Both the protein levels and the DNA binding activity of the two virus-induced cellular transcription factors Sp1 and NF-kappaB were found to be markedly reduced in the presence of artesunate. We also analyzed the cellular signaling kinase phosphoinositide 3-kinase, required for the activation of factors such as Sp1 and NF-kappaB in infected fibroblasts. The phosphorylation of two downstream effectors of phosphoinositide 3-kinase, Akt and p70S6K, was markedly inhibited in the presence of artesunate. Thus, artesunate possesses attractive antiviral characteristics which are suggestively based on the interference with essential steps in the host cell kinase cascades.