Human embryonic stem cells (hESCs) have the potential to differentiate to diverse cell types. This ability endows hESCs with promise for the development of novel therapeutics, as well as promise for the development of a rigorous genetic system to probe human gene function. However, in spite of the impending utility of hESCs for clinical and basic applications, little is known about their fundamental properties. Recent reports have documented transcriptional profiles of mouse embryonic stem cells (mESCs), adult stem cells and a single hESC line, H9. To date, however, the transcriptional profiles of independently-derived hESC lines have not been compared. In order to examine the similarities and differences in multiple hESC lines, we compared gene expression profiles of the HSF-1, HSF-6 and H9 lines. We found that the majority of genes examined were expressed in all three cell lines. However, we also observed that each line possessed a unique expression signature; the expression of many genes was limited to just one or two hESC lines. We suggest that the observed differences in gene expression between independently-derived hESC lines may reflect inherent differences in the initial culture of each line and/or the underlying genetics of the embryos from which the lines were derived.