Objective: To investigate the fertilization ability, chromatin structure, and DNA integrity of spermatozoa after being cryopreserved within an empty zona pellucida (ZP). Design: Prospective study. Setting: Reproductive research center in a university-affiliated hospital. Patient(s): Normozoospermic patients. Intervention(s): Spermatozoa were cryopreserved within the ZP or with traditional methods. Main Outcome Measure(s): The sperm recovery rate, sperm motility, fertilization ability, chromatin structure, and DNA integrity were assessed before and after cryopreservation. Result(s): Significantly higher sperm recovery rate was identified for the spermatozoa cryopreserved within the ZP than those cryopreserved with traditional methods, but the motility recovery was similar. Frozen-thawed samples showed increased damage to the sperm chromatin and DNA, which were assessed by acridine orange test (AO) and terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling assay; however, no difference of chromatin and DNA integrity was observed for spermatozoa cryopreserved within the ZP or with traditional methods. In addition, the fertilization ability, as assessed by injecting spermatozoa into hamster oocytes, was similar for spermatozoa cryopreserved with different cryopreservation methods. Conclusion(s): Cryopreservation of spermatozoa within an empty ZP results in higher sperm recovery rate, and the post-thaw sperm functions of spermatozoa cryopreserved within the ZP are comparable with spermatozoa cryopreserved with traditional methods. © 2009 American Society for Reproductive Medicine.