We have identified what is apparently a distinct type of astrocyte in primary cultures from several regions of the neonatal rat CNS. These cells express GD3 ganglioside for long periods in vitro, and are GFAP+, but do not express the oligodendrocyte antigens O4 or galactocerebroside (GC). The majority, but not all, are A2B5+. The cells grow in a flat, highly spread morphology with many thin cytoplasmic processes. Gene transfer with a replication-deficient retrovirus combined with immunostaining for astro- and oligodendroglial markers (antibodies to GFAP, GD3 ganglioside, GC, and the A2B5 and O4 antibodies) demonstrated that in the neonatal rat CNS cultures these cells are clonally separate from oligodendrocytes and from the majority of (GD3-) astrocytes. The clonal analysis suggests a distinct progenitor cell and a distinct developmental sequence for these astrocytes.