Activation of cytosolic phospholipase A2 (cPLA2) is a prerequisite for the formation of the transcription factor complex interferon-stimulated gene factor 3 (ISGF3) in response to interferon-α (IFN-α). Here we show that p38 mitogen-activated protein kinase (MAPK), an activator of cPLA2, is essential for both IFN-α and IFN-γ signalling. SB203580, a specific inhibitor of p38, was found to inhibit ISGF3 formation but had no apparent effects on signal transducer and activator of transcription (STAT)1 homodimer formation. Regardless of this, the antiviral activities of both IFN-α and IFN-γ were attenuated by SB203580. Treatment with either IFN led to rapid and transient activation of p38. Both IFNs induced STAT1 Ser727 phosphorylation, which was inhibited by SB203580 but not by an extracellular signal related kinase (ERK)1/2 inhibitor (PD98059). In an inducible 3T3-L1 clone, expression of dominant-negative p38 led to defective STAT1 serine phosphorylation and diminished IFN-γ-mediated protection against viral killing. Reporter activity mediated by ISGF3 or STAT1 homodimer was diminished by SB203580 and enhanced by a constitutively active mutant of MKK6, the upstream activator of p38. Therefore, p38 plays a key role in the serine phosphorylation of STAT1 and transcriptional changes induced by both IFNs.