Our objectives in this study were to investigate the relationship between ATP production (oxidative phosphorylation and glycolysis) and fertility of bovine spermatozoa; determine the proportion of oxygen consumption devoted to proton leak and that due to nonmitochondrial processes; and discover if freeze/thawing affects sperm oxygen consumption. Oxygen consumption of bovine spermatozoa was measured using a standard Clark electrode and, for the first time, in an Oxygen Biosensor System (OBS). Total ATP formation by bovine spermatozoa was calculated from the oxygen consumption and lactate production (glycolysis) by the same spermatozoa sample. ATP production varied from 1.99 to 8.09 µmol ATP/ 10⁸ spermatozoa/ hour; glycolysis accounted for 16-38% of ATP. Non-mitochondrial oxygen consumption could not be detected in bovine spermatozoa using these methods. A significant proportion, 16-43%, of oxygen consumption was insensitive to oligomycin, and was due to "proton leak." There was no significant difference between oxygen consumption of frozen/thawed and fresh spermatozoa for two of the three bulls tested. However, oxygen consumption of frozen/thawed spermatozoa was significantly higher (P<0.05) than fresh spermatozoa for the third bull. When ZO2 of frozen/thawed spermatozoa from 20 bulls was compared with their 49 day non-return rates (NRRs), oxygen consumption was correlated positively with NRR, i.e. fresh spermatozoa with a higher ZO₂ were more fertile. Moreover, total ATP production correlated with NNR better than ZO₂. Bulls with a lower NRR produce spermatozoa which are susceptible to damage during the freeze/thawing process, causing an increase in ZO₂; possibly due to mitochondrial membrane damage resulting 1 in more energy being expended in maintaining the proton gradient, or capacitation-like changes causing hyperactivation. Oxygen consumption measured in the OBS may be useful in assessing bovine sperm fertility.