Abnormal growth factor signaling is implicated in the pathogenesis of gliomas. The extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway is a likely target, linking receptor tyrosine kinase activation to downstream serine/threonine phosphorylation events regulating proliferation and differentiation. Signaling within heterogeneous cell populations of gliomas cannot be adequately assessed by traditional biochemical enzyme assays. Immunohistochemical detection of doubly phosphorylated (activated) ERK/MAPK permitted visualization of spatially discrete cellular patterns of ERK/MAPK activation, compared with the relatively uniform expression of total ERK/MAPK protein. The astrocytic tumors, regardless of grade, had the highest overall degree of enzyme activation, whereas oligodendrogliomas had the least. Anaplastic progression in oligodendrogliomas resulted in a larger number of cells with active ERK/MAPK. Within glioblastomas, microvascular hyperplasia and necrosis were associated with ERK/MAPK activation in adjacent tumor cells. In addition to spatial patterns of intratumor paracrine signaling, a possible cell-cycle-associated regulation was detected: mitotic and actively cycling tumor cells showed diminished activation relative to cells in G0. Although ERK/MAPK activation was not restricted to neoplastic glia, consistent patterns of selective activation in tumor cells suggests that sustained activation may contribute to the neoplastic glial phenotype.