Schwann cells (SCs) play an important role in the pathogenesis of peripheral nerve diseases and represent a potential target for development of therapies. However, use of primary human SCs (hSCs) for in vitro models is limited because these cells are difficult to prepare and maintain in high yield and purity under common cell culture conditions. To circumvent this obstacle, we immortalized primary human fetal SCs using the SV40 large T-antigen and human telomerase reverse transcriptase expression vectors. After cloning, selection, and purification, we evaluated several immortalized SC lines for their ability to express extracellular matrix (ECM) molecules and myelinate embryonic rat sensory axons. In addition, we established a gene expression profile and explored their sensitivity to oxidative stress in a simple in vitro assay. Immortalized hSC clones expressed common glial markers and a broad variety of growth factors, receptors, and ECM molecules as determined by immunocytochemistry, microarray, and quantitative reverse transcription-polymerase chain reaction. In neuron-SC co-cultures, these cells were able to myelinate rat dorsal root ganglia neurons, although their effectiveness was lower in comparison to primary rat SCs. In toxicity assays, immortalized hSCs remain susceptible to oxidative stress induced by H(2)O(2). This study shows that, using specific immortalization techniques, it is possible to establish hSC lines that retain characteristics of typical primary hSCs. These cells are particularly useful for drug screening and studies aimed at disease mechanisms involving SCs.