The objectives of this study were to evaluate the hematopoietic activity of peripheral blood stem cells (PBSCs) harvested from patients with advanced germ cell tumors after long-term cryopreservation in a mechanical freezer at -80 degrees C. Forty-one patients with advanced tumors were enrolled in this study. Flow cytometric analysis was performed to determine the total cell viability and the viability of CD34 positive cells of PBSCs stored between January 1995 and December 2001. The 41 patients were divided into two groups according to the duration of cryopreservation. In the 21 patients in whom the duration of storage was greater than 5 years, the total cell number did not change significantly before and after long-term cryopreservation (p=0.75). The percentage of viable cells and the total number of viable cells was significantly decreased after long-term cryopreservation (p<0.001 and p<0.05, respectively). However, the percentage of CD34 positive cells was significantly increased to 2.34% from 1.85% (p<0.05), resulting in non-statistically significant change in the total viable CD34 positive cells (p=0.98). A similar relation was found in the 20 patients whose storage duration was less than 2 years, although a significant change was found only in the percentage of viable cells. Moreover, pre-PBSCH factors, including age, cumulative dosage of cisplatin and the International Germ Cell Cancer Collaborative Group classification at the diagnosis did not affect the viable CD34 positive cell count after long-term cryopreservation. In conclusion, the present results suggest that CD34 positive cells could be successfully cryopreserved in a mechanical freezer at -80 degrees C for more than 5 years with no relation to pre-PBSCH factors and efficacy of PBSCH itself.