In a number of pathological states of the brain, the activation of the inducible nitric oxide synthase (iNOS) plays a major role. Interleukin (IL)-1beta is believed to be an essential factor in the induction of iNOS. However, little is known about the cascade of events culminating in iNOS expression in vivo. To identify the morphological as well as temporal relationship of lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma) -induced microglial iNOS- and IL-1beta expression, a mixture of LPS and IFN-gamma was injected into the rat hippocampus. IL-1beta immunoreactivity was detected as early as 3 hr following surgery in ramified microglia in the lesioned hippocampus and in distal cortical layers adjacent to the pia mater. By 12 hr post-injection, IL-1beta immunoreactive, ramified microglia with swollen processes were widely distributed throughout hippocampal and neocortical areas, and staining was observed up to 48 hr after treatment. In contrast, iNOS immunostaining was seen in activated amoeboid microglia/macrophages in the ipsilateral hippocampus and around blood vessels but not earlier than 12 hr post-surgery. The temporal pattern of iNOS and IL-1beta expression corresponded to newly induced transcriptional activity as revealed by RT-PCR. Activation of NF-kappaB was restricted to brain regions in which IL-1beta was expressed and was detected both in microglia and astrocytes. A number of LPS/IFN-gamma-stimulated, IL-1beta-expressing microglia exhibited co-staining for activated NF-kappaB. The finding that IL-1beta precedes iNOS expression is consistant with a role of IL-1beta in the intercellular signaling events leading to microglial iNOS-induction. Co-localization of IL-1beta and NF-kappaB suggests an association between IL-1beta and NF-kappaB induction.