OBJECTIVE In vivo effects of tritium contamination are poorly documented. Here, we study the effects of tritiated Thymidine ([(3)H] Thymidine) or tritiated water (HTO) contamination on the biological properties of hematopoietic stem cells (HSC). METHODS Mouse HSC were contaminated with concentrations of [(3)H] Thymidine ranging from 0.37-37.03 kBq/ml or of HTO ranging from 5-50 kBq/ml. The biological properties of contaminated HSC were studied in vitro after HTO contamination and in vitro and in vivo after [(3)H] Thymidine contamination. RESULTS Proliferation, viability and double-strand breaks were dependent on [(3)H] Thymidine or HTO concentrations used for contamination but in vitro myeloid differentiation of HSC was not affected by [(3)H] Thymidine contamination. [(3)H] Thymidine contaminated HSC showed a compromised long-term capacity of hematopoietic reconstitution and competition experiments showed an up to two-fold decreased capacity of contaminated HSC to reconstitute hematopoiesis. These defects were not due to impaired homing in bone marrow but to an initial decreased proliferation rate of HSC. CONCLUSIONS These results indicate that contaminations of HSC with doses of tritium that do not result in cell death, induce short-term effects on proliferation and cell cycle and long-term effects on hematopoietic reconstitution capacity of contaminated HSC.