OBJECTIVE:To evaluate sperm cryopreservation-induced injuries using sperm plasma membrane protein P34H and alpha-tubulin as two different subcellular compartment markers. DESIGN:Prospective experimental study. SETTING:Academic hospital research center and fertility clinic. PATIENT(S):Semen samples obtained from healthy donors attending the fertility clinic. Sperm samples were either directly processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot experiments (control group) or cryopreserved in liquid nitrogen for different periods of time before being analyzed. INTERVENTION(S):SDS-PAGE, Western blotting, and densitometric quantification of P34H and alpha-tubulin before and after cryopreservation. MAIN OUTCOME MEASURE(S):Changes in protein quantification between the different groups as a result of sperm cryopreservation. RESULT(S):In the 31 sperm samples processed for P34H evaluation, a 50% decrease is observed after sperm cryopreservation as compared to the control group. The alpha-tubulin immunoblotting of 41 sperm samples revealed a 200% increase in the protein detection in the group of cryopreserved sperm as compared to the control group. Contrary to the P34H detection, this change in alpha-tubulin immunodetected levels appears to be related to the cryopreservation period as it increases during storage. CONCLUSION(S):These findings indicate that cryopreservation of human semen induces damages at different cellular levels. Moreover, some cryoinjuries are immediate although others seem to take place over time stored in liquid nitrogen.