We have investigated beta interferon (IFN-beta) and IFN-alpha4 gene expression and activation of related transcription factors in mouse cytomegalovirus (MCMV)-infected fibroblasts. mRNA analysis demonstrated an initial phase of IFN gene induction upon MCMV infection, which was followed by a sustained MCMV-mediated simultaneous downregulation of IFN-beta and IFN-alpha4 gene expression. The induction of IFN transcription resulted from the activation of the components of the IFN-beta enhanceosome, i.e. IFN regulatory factor (IRF) 3, nuclear factor (NF)-kappaB, activating transcription factor (ATF)-2 and c-Jun. Activation of the transcription factors occurred rapidly and in a sequential order upon infection, but only lasted a while. As a consequence, IFN-alpha/beta gene expression became undetectable 6 h post-infection and throughout the MCMV replication cycle. This effect is based on an active interference since restimulation of IFN gene induction by further external stimuli (e.g. Sendai virus infection) was completely abolished. This inhibition required MCMV gene expression and was not observed in cells infected with UV-inactivated MCMV virions. The efficiency of inhibition is achieved by a concerted blockade of IkappaBalpha degradation and a lack of nuclear accumulation of IRF3 and ATF-2/c-Jun. Using an MCMV mutant lacking pM27, a signal transducer and activator of transcription (STAT) 2-specific inhibitor of Jak/STAT signalling, we found that the initial phase of IFN induction and the subsequent inhibition does not depend on the positive-IFN feedback loop. Our findings indicate that the MCMV-mediated downregulation of IFN transcription in fibroblasts relies on a large arsenal of inhibitory mechanisms targeting each pathway that contributes to the multiprotein enhanceosome complex.