Normal human esophageal autopsy tissue was explanted in serum-free medium. The epithelial outgrowths were subcultured and then transfected by strontium phosphate coprecipitation with plasmid pRSV-T consisting of the RSV-LTR promoter and the sequence encoding the simian virus 40 large T-antigen. The transfected cells, but not the sham-transfected controls, formed multilayered colonies within 3–4 weeks, after which the colonies were transferred and cell strains (HE-451 and HE-457) developed. Both cell strains grew exponentially for 8–10 weeks and then senesced. After a “crisis” of 6–8 months, growth resumed in isolated colonies. One line, HET-1A from HE-457, was developed and has now undergone more than 250 population doublings. This line has retained epithelial morphology, stains positively for cytokeratins and the simian virus 40 T-antigen gene by immunofluorescence, and has remained non-tumorigenic in athymic, nude mice for more than 12 months. Karyotypic analysis by Giemsa banding has shown that HET-1A is hypodiploid (34–40 chromosomes). Growth factor studies have shown that HET-1A is stimulated by Ca2+, and inhibited by fetal bovine serum, transforming growth factor-β1, and transforming growth factor-β2. This serum-free immortalized esophageal cell system will be useful for investigating the action of putative esophageal carcinogens.