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The objective of this flow cytometric study was to examine plasma membrane integrity, mitochondrial membrane potential (MMP) and the degree of DNA fragmentation of cryopreserved bovine sperm immediately (0 h) and 3 h after thawing and to compare the results with each other and with the fertility of bulls. Cryopreserved spermatozoa from 4 consecutive ejaculates of 20 bulls were examined. Percentages of plasma membrane intact sperm (PMI) and sperm showing a high MMP (HMMP), respectively, were determined by the SYBR14/PI- and the JC-1 assays. DNA fragmentation was analysed by the standard deviation of the DNA fragmentation index (SD-DFI) and the percentage of sperm with a high degree of DNA fragmentation (%DFI) by using SCSATM. The mean non-return rate on day 56 (NRR 56) ranged from 63.7% to 78.0% (mean ± SD: 71.8% ± 3.7%). Mean values for PMI and HMMP decreased from 37.4% ± 6.8% to 31.2% ± 6.1% and from 38.8% ± 7.1% to 23.8% ± 7.7% respectively. SD-DFI increased from 56.9% ± 8.0% to 69.0% ± 12.9% and %DFI from 6.4% ± 2.5% to 12.4% ± 5.8%. The correlation between PMI 0 h and HMMP 0 h (r = 0.95; p < 0.0001) was higher (p < 0.05) than that between PMI 3 h and HMMP 3 h (r = 0.88; p < 0.0001). %DFI 0 h was neither related to PMI 0 h nor to HMMP 0 h (p > 0.05), nor was there a correlation (p > 0.05) between DFI 3 h and PMI 3 h; but %DFI 3 h and HMMP 3 h were significantly correlated (r = −0.31; p < 0.05). SD-DFI and %DFI 3 h were the only parameters related to NRR 56 (r = −0.58; p < 0.05). In conclusion, plasma membranes and mitochondria are similarly affected by the freezing and thawing process, but not during the incubation period after thawing.