Carbonic anhydrase (CA) was studied in primary monolayer cultures from neonatal rat cerebral hemispheres with both immunocytochemical and biochemical techniques. In such cultures, which consist predominantly of astrocytes, immunocytochemical staining for CA using antibody raised against the type II enzyme from rat erythrocytes resulted in positive staining of the flat, glial fibrillary acidic protein-positive, astrocytic monolayer. Smaller, process-bearing, round cells that grew on top of the astrocytes stained intensely for CA. We estimated that these cells represented 1% or less of the total cells in the cultures, and they have been identified by others as oligodendrocytes. The intensity of the staining of astrocytes for CA could be increased to that observed in oligodendrocytes when the astrocytes were made to round up and form processes by treatment with 2',3'-dibutyryl cyclic AMP. Enzymatic assays showed that CA activity of the cultures after 3 weeks of growth was 2.5- to 5-fold less than that found for cerebral homogenates from perfused 3-week-old rat brains. However, both activities were totally inhibited by acetazolamide with an I50 of 10(-8) M, confirming that both rat brain and the astrocyte cultures possess the high-activity type II enzyme. CA-II activity was unaffected by treatment of the cultures with a method reported to remove oligodendrocytes. Thus, the immunocytochemical and biochemical studies reported here demonstrate that astroglial cells in primary cultures from neonatal rat brain contain CA-II.