It has been postulated that production of TNF-α is central to the pathogenesis of septic shock induced by group B Streptococcus (GBS). In vitro studies using human cord blood monocytes have demonstrated that GBS induces TNF-α secretion, but little is known about the intracellular signaling pathways of TNF-α induction. In this report we show that heat-killed serotype III GBS induces host cell signal transduction pathways that lead to activation of the transcription factors NF-κB and AP-1. Using adenoviral transfer of IκBα (IκBα overexpression), the production of TNF-α induced by whole GBS was inhibited by only 20%. We also show that the p38 mitogen- activated protein kinase (MAPK) pathway is involved in GBS-induced TNF-α secretion, because TNF-α protein and mRNA levels in the presence of a specific inhibitor of p38 MAPK, SB 202190, were dramatically diminished. EMSAs showed that SB 202190 inhibited GBS-induced AP-1 activation, but had no effect on NF-κB-DNA binding activity. These results indicate that both NF- κB and AP-1 (via p38 MAPK) are involved in the regulation of TNF-α production in GBS-stimulated neonatal monocytes. Therefore, disrupting the signal transduction pathways induced by GBS has the potential to attenuate the production of immune response mediators, thereby halting or possibly reversing the course of this potentially fatal disease.