The glial cells that ensheath olfactory axons are referred to as ensheathing cells. In vivo, these non-myelinating glial cells express a mixture of astrocyte-specific and Schwann cell-specific phenotypic features with the former cellular phenotype predominating, but in vitro can assemble a myelin sheath when co-cultured with dorsal root ganglion neurons. Thus, certain in vitro conditions induce ensheathing cells to express a phenotype more like that of a myelinating Schwann cell. The present study addresses whether ensheathing cells will express a myelinating phenotype in neuron-free cultures when fed for 1 to 5 weeks with media shown to promote the growth and differentiation of oligodendrocyte progenitor cells. The ensheathing cell cultures were initiated using the nerve fiber layers (NFL) of rat olfactory bulb primordia. Oligodendrocyte cultures were established from newborn rat neopallium and from the tissue that remained after removing the NFL from the developing olfactory bulb (i.e., the OB-NFL). The cultures were double-labelled with rabbit polyclonal antibodies to S100 or glial fibrillary acidic protein in combination with the mouse monoclonal antibodies O4, BRD1 [anti-galactocerebroside (anti-GAL-C)], and anti-myelin basic protein (MBP). In some experiments the ensheathing cells were labelled with PKH26 prior to being co-cultured with oligodendrocytes of the OB-NFL. None of the media induced ensheathing cells to express either GAL-C or MBP. However, when 0.5 mM dibutyrylcyclic-AMP (dBcAMP) was added to the medium, ensheathing cells became GAL-C + ve, but remained MBP-ve. The molecular mechanisms that regulate the expression of a myelinating phenotype by ensheathing cells appear to be different from those that operate in oligodendrocytes.