Several laboratories have begun evaluating human ES (hES) cell lines; however, direct comparisons between different hES cell lines have not been performed. We have characterized the properties of four human cell lines maintained in feeder-free culture conditions. Quantitative assessment of surface markers, microarray analysis of gene expression patterns, expression of SOX-2, UTF-1, Rex-1, OCT3/4, CRIPTO, and telomerase activity demonstrated similar patterns in all hES cell lines examined. Undifferentiated hES cells do not respond to neurotransmitters such as acetylcholine, glutamate, and gamma-aminobutyric acid. In addition, the undifferentiated hES cells possess gap junctions. Although similarities in marker expression were observed, allotyping showed that all four lines have a distinct HLA profile, predicting differences in transplantation responses. These data provide the first detailed comparison of different hES cell lines and demonstrate remarkable similarities among lines maintained in identical culture conditions. Developmental Dynamics 229:243–258, 2004. © 2003 Wiley-Liss, Inc.