The objective of this study was to determine whether vimentin expression by process-bearing astroglia and oligodendroglia cultured from neonatal rat cerebral cortex resembled that in brain where vimentin is common in immature astroglia and a few subpopulations of mature astroglia, but is absent in oligodendroglia. Vimentin expression was detected by immunofluorescence microscopy using a monoclonal antibody (V9) against porcine lens vimentin in combination with either antiserum against the astroglial marker, glial fibrillary acidic protein (GFAP), or with antiserum against the oligodendroglial marker, galactocerebroside (GC). Specificity of the antivimentin antibody was indicated on immunoblots of process-bearing cell proteins separated by two-dimensional polyacrylamide gel electrophoresis. Enrichment of cultures for either GFAP+ astroglia or GC+ oligodendroglia was achieved by supplementation of the culture medium with fetal calf serum at 10% or 0.5%, respectively. Process-bearing cells maintained in 10% serum exhibited heterogeneity in their expression of GFAP and vimentin. Approximately half of the cells were GFAP+/vimentin+ throughout the 2-week culture period examined. GFAP+/vimentin- cells were a minor population at early times (3-4 days) in culture, but accounted for 40% of process-bearing cells after 2 weeks. Cultures maintained in reduced (0.5%) serum and stained for GC and vimentin also exhibited heterogeneity. Both GC+/vimentin+ and GC+/vimentin- cells were observed, with vimentin+ cells composing two-thirds and one-half of the GC+ population after 3 and 6 days, respectively, in reduced serum. The high incidence of vimentin expression by process-bearing astroglia and oligodendroglia suggests that these cultures contain glia in a relatively early stage of development.(ABSTRACT TRUNCATED AT 250 WORDS)