The role of bacterial invasion and subsequent intracellular metabolism or replication, or both, in the induction of interferon (IFN) production in primary cultures of murine embryo fibroblasts (MEFs) was examined. IFN production appeared to be dependent upon bacterial invasion. MEFs that were challenged with Shigella flexneri cultured at 30 degrees C to inhibit the temperature-dependent virulence gene expression that is essential for invasion failed to produce IFN. Furthermore, inhibition of S. flexneri invasion by pretreatment of MEFs with cytochalasin B resulted in a reduction in IFN production. Intracellular bacterial residence alone, however, was not sufficient for the induction of IFN production since an avirulent isogenic variant of S. flexneri which invades but fails to grow intracellularly did not induce IFN production. In fact, the blocking of bacterial RNA synthesis immediately after cellular uptake of S. flexneri by rifampin inhibited IFN production by MEFs. Transfer of the invasion-encoding plasmid to a noninvasive Escherichia coli strain conferred upon the bacteria the ability to invade MEFs and induce IFN production. These results suggest that the induction of IFN production in S. flexneri-infected fibroblasts requires bacterial invasion and intracellular bacterial metabolism or replication, or both.