The use of immortalized cells has been instrumental as a tool with which to study gene regulation. However, it is crucial to understand the status of a given cell line, especially when investigating the regulation of genes whose expression is developmentally regulated. Several immortalized cell lines have been derived from primary cultures of mouse oligodendrocytes. Two such cell lines, N20.1 and Oli-neu, were characterized here in terms of their relative expression of myelin genes at both the mRNA level and the protein level. Analysis of the splice isoforms expressed by the myelin proteolipid protein (Plp1), myelin basic protein (Mbp), and 2′,3′-cyclic nucleotide 3′-phosphodiesterase (Cnp) genes, along with the relative amount of protein expressed by these genes, suggests that the cell lines are representative of immature oligodendrocytes, although Oli-neu cells appear to be farther along the differentiation pathway compared with N20.1 cells. Previous studies have shown that the developmental increase in Plp1 gene expression that occurs during the active myelination period is governed by transcription regulatory elements present within the first intron. The responsiveness of one of these elements, the so-called antisilencer/enhancer (ASE), was investigated in both cell lines. Results presented here suggest that the ASE has a much more potent effect in Oli-neu cells. Thus, the two cell lines appear to be at different stages and will be useful as a means to study transcription regulatory elements whose influence changes during development. © 2011 Wiley-Liss, Inc.