The multiplication of the human neurotropic polyomavirus JC virus (JCV) is regulated by cell membrane receptors and nuclear transcription factors. Signaling pathways also play a role in determining the extent to which JCV can productively infect cells. These data show that constitutively active MEK1 protein (CA-MEK1), overexpressed in cultures of human glia, supports a substantial increase in late JCV protein (Vp-1) synthesis. The specificity of this pathway was indicated by no significant enhancement of JCV multiplication through activation of other components of mitogen-activated protein kinase pathways such as p38, Jun N-terminal protein kinase, and protein kinase A. Further evidence supporting the importance of signaling in JCV infection came from addition of transforming growth factor beta1 (TGF-beta1), which stimulated a 200% increase of Vp-1 expression. Specific MEK1/2 inhibitors, flavenoid PD98059 and U0126, decreased the basal and TGF-beta1-stimulated Vp-1 expression by 95% or more. TGF-beta1 is known to phosphorylate/activate Smad DNA binding proteins that could subsequently bind or increase binding to JCV promoter sequences, linking the effects of signaling with JCV transcriptional regulation. The effectiveness with which MEK1/2 inhibitors block JCV multiplication provides insight that may contribute to development of compounds directed against JCV.