A high-throughput screening (HTS) assay for inhibitors of nitric oxide (NO) production by activated microglia was developed and used to compare the relative activities of various anti-inflammatory compounds and cell-permeable protein kinase inhibitors. BV-2 cells, an immortalized line that retains phenotypic features of microglia and produces NO in response to lipopolysaccharide (LPS), were used in the activation paradigm for the HTS assay. A characteristic feature of the compounds that were the most potent dose-dependent inhibitors of NO production is their ability to modulate serine/threonine protein kinases. The anti-inflammatory compound K252a, an inhibitor of calmodulin (CaM)-regulated protein kinases, had one of the highest potencies in the assay. Other classes of kinase inhibitors, including the protein kinase A inhibitor H-89, the mitogen activated protein kinase inhibitors PD98059 and SB203580, and the tyrosine kinase inhibitor genistein, were less potent and efficacious than K252a or the general serine/threonine/tyrosine kinase inhibitor staurosporine. K252a suppresses production of the inducible nitric-oxide synthase (iNOS). The inhibitory effect of K252a is not due to cell toxicity and does not correlate with inhibition of NFkappaB nuclear translocation. The mechanism of action appears to involve inhibition of phosphorylation of the transcription factor CREB, a protein whose activity is modulated by phosphorylation by CaM-dependent protein kinases. These data suggest that signal transduction pathways mediated by CaM-dependent protein kinases warrant future study as potential drug discovery targets.