BACKGROUND: Embryonic stem cells (ESC) maintain their 'stemness' by self-renewal. However, the molecular mechanisms underlying self-renewal of human embryonic stem cells (hESC) remain to be elucidated. In this study, expression profiles of the molecules of developmentally important signalling pathways were investigated to better understand the relationships of the signalling pathways for self-renewal in hESC. METHODS: Two human ESC lines were cultured on mouse embryonic fibroblast (MEF) feeder cells. Gene expression was analysed by RT-PCR, real-time RT-PCR and Western blotting. RESULTS: In the bone morphogenetic protein (BMP4), transforming growth factor (TGF-beta) and fibroblast growth factor (FGF4) signalling pathways, ligands and antagonists were highly expressed in hESC compared with human embryoid body (hEB). Human ESC showed abundant transcripts of intracellular molecules in the Wnt, Hh and Notch signalling pathways. No difference was detected in the expression level of the JAK/STAT signalling molecules between hESC and hEB. Western blot analysis showed that the transcriptional levels of the signalling molecules in hESC were consistent with translational levels. From the real-time PCR analysis, expression levels of some genes, such as Oct3/4, Nodal and beta-catenin, were different between two hESC lines. CONCLUSION: The self-renewal of hESC is probably maintained by coordinated regulation of signalling-specific molecules and in a signalling-specific manner.