The pentaspan protein CD133 (Prominin-1) is part of the signature of tumour-initiating cells for various cancer entities. The aim of the present study was to investigate the impact of ectopic CD133 expression on tumourigenic properties of otherwise CD133-negative, non-tumourigenic cells in vitro and in vivo. CD133 was stably transfected into human embryonic kidney 293 (HEK293) which was then sorted for the expression of CD133. The effects of CD133 on cell proliferation were assessed upon standard cell counting of sorted cells at various time points. Severe combined immunodeficient (SCID) mice (n = 30) were injected with HEK293 CD133(high) and CD133(low) transfectants (5 × 10(3), 1 × 10(5), or 5 × 10(6) cells per injection). The expression of CD133, Ki67, CD44s, CD44v6, and EpCAM was analysed upon immunohistochemical staining of cryosections with specific antibodies. In vitro, ectopic expression of CD133 did influence neither cell proliferation nor cell cycle distribution of otherwise CD133-negative HEK293 cells. However, CD133(high) cells generated tumours in vivo in SCID mice with at least 1,000-fold increased frequency compared to CD133(low) cells. Tumour load was also significantly increased in CD133(high) cells as compared to those tumours formed by high numbers of CD133(low) cells. Immunohistochemistry stainings disclosed no changes in Ki67, CD44s, CD44v6, or EpCAM once tumours were formed by either cell type. CD133 induces tumour-initiating properties in HEK293 cells in vivo and is potentially involved in the regulation of tumourigenicity. Future research will aim at the elucidation of molecular mechanisms of CD133-induced tumourigenicity.