Interferon (IFN)-alpha affects the growth, differentiation and function of various cell types by transducing regulatory signals through the Janus tyrosine kinase/signal transducers of activation and transcription (Jak/STAT) pathway. The signalling pathways employing the mitogen-activated ERK-activating kinase (MEK) and the extracellular-regulated kinase (ERK) are critical in growth factors signalling. Engagement of the receptors, and subsequent stimulation of Ras and Raf, initiates a phosphorylative cascade leading to activation of several proteins among which MEK and ERK play a central role in routing signals critical in controlling cell development, activation and proliferation. We demonstrate here that 24-48 h following treatment of transformed T- and monocytoid cell lines with recombinant human IFN-alpha2b both the phosphorylation and activity of MEK1 and its substrates ERK1/2 were reduced. In contrast, the activities of the upstream molecules Ras and Raf-1 were not affected. No effect on MEK/ERK activity was observed upon short-term exposure (1-30 min) to IFN. The anti-proliferative effect of IFN-alpha was increased by the addition in the culture medium of a specific inhibitor of MEK, namely PD98059. In conclusion, our results indicate that IFN-alpha regulates the activity of the MEK/ERK pathway and consequently modulates cellular proliferation through a Ras/Raf-independent mechanism. Targeting the MEK/ERK pathway may strengthen the IFN-mediated anti-cancer effect.