BACKGROUND: The aim of this study was to characterize human embryonic stem (ES) cells at the molecular level by performing large-scale complementary DNA (cDNA) analysis using DNA micro-arrays. METHODS: The transcription profile of human ES cells was determined by comparing it to 2, 10 and 30-day old embryoid bodies (EBs) using Affymetrix Genechip human micro-arrays (U133). RESULTS: According to this analysis we demonstrate that two human ES cell lines are more close to each other than to their differentiated derivatives. We also show the spectrum of cytokine receptors that they express, and demonstrate the presence of five genes that are highly specific to human ES cells and to germ cells. Moreover, by profiling different stages in the differentiation of human embryoid bodies, we illustrate the clustering of five sets of temporally expressed genes, which could be related to the sequential stages of embryonic development. Among them are known genes that are involved in early pattern formation. CONCLUSIONS: The present study provides a molecular basis for the identity of human ES cells and demonstrates that during their in vitro differentiation they express embryonic specific genes in a stage specific manner.