It has long been recognized that T cells in the cerebrospinal fluid (CSF) and other inflammatory compartments cannot be stimulated by mitogen and the reason for this has remained unknown. This question was investigated using mononuclear cells (MNC) isolated from the CSF of subjects with multiple sclerosis and other inflammatory brain diseases which predominantly express the CD4 and CDw29 but not CD45RA determinants. CSF and blood cells were stimulated by either the CD3/T cell receptor complex, the CD2 activation pathway, calcium ionophore, or an activator of protein kinase C, phorbol myristate acetate (PMA). CSF MNC proliferated less than blood MNC following stimulation by phytohemagglutinin in subjects with inflammation in the CSF, but not in subjects with non-inflammatory CNS diseases. Moreover, CSF MNC were not induced to proliferate through stimulation of the CD2 pathway by anti-T11(2) + anti-T11(3) monoclonal antibodies (mAb). This was not due to defects in either interleukin 2 receptors, interleukin 2 secretion, or to T cell pre-activation in vivo. Instead, the refractory activation state of inflammatory CSF T cells was corrected by PMA. That CSF contains predominantly CD4+CDw29+CD45RA- cells suggests that PMA may be co-stimulatory with anti-CD2 mAb to activate this population of T cells. This was confirmed in experiments with sorted T cells from normal subjects. These data suggest that the inability of mitogens or anti-CD2 mAb to stimulate inflammatory CSF T cells, which can be corrected by an inducer of protein kinase C, is related to the relative absence of CD4+CD45RA+ cells in the CSF. Alterations of protein kinase C and protein phosphorylation may exist in inflammatory T cell populations that regulate the immune response.