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Positive selected haematopoietic stem cells are increasingly used for allogeneic transplantation with the CD34 antigen employed in most separation techniques. However, the recently described pentaspan molecule CD133 appears to be a marker of more primitive haematopoietic progenitors. Here we report our experience with a new CD133-based selection method in 10 paediatric patients with matched unrelated (n = 2) or mismatched-related donors (n = 8). These patients received a combination of stem cells (median = 29.3 x 10(6)/kg), selected with either anti-CD34 or anti-CD133 coated microbeads. The proportion of CD133+ selected cells was gradually increased from patient to patient from 10% to 100%. Comparison of CD133+ and CD34+ separation procedures revealed similar purity and recovery of target populations but a lower depletion of T cells by CD133+ selection (3.7 log vs. 4.1 log, P < 0.001). Both separation procedures produced >90% CD34+/CD133+ double positive target cells. Engraftment occurred in all patients (sustained primary, n = 8; after reconditioning, n = 2). No primary acute graft versus host disease (GvHD) >/= grade II or chronic GvHD was observed. The patients showed a rapid platelet recovery (median time to independence from substitution = 13.5 d), whereas T cell regeneration was variable. Five patients are alive with a median follow-up of 10 months. Our data demonstrates the feasibility of CD133+ selection for transplantation from alternative donors and encourages further trials with total CD133+ separated grafts.