Protein tyrosine phosphorylation plays a regulatory role in a multitude of physiological processes in sperm. Changes in protein tyrosine phosphorylation, viability, and motility were studied as a function of extended incubation of bovine sperm in vitro at ambient temperature (18–20°C). Fresh ejaculates were incubated after dilution for 8 days. On Days 0, 2, 5, and 8, an aliquot of sperm was incubated with or without theophylline at 37°C for 30 min prior to assessing sperm viability, motility, and tyrosine phosphorylation of soluble and whole-cell proteins. There was a time-dependent decline in sperm motility, which was to some extent reversed by incubation with theophylline. The sum of the phosphotyrosine signal from two soluble proteins (M_r 67 000 and 36 000) declined with incubation time in both theophylline-treated and untreated sperm. There were major differences in the pattern of tyrosine phosphorylation during incubation between ejaculates from different bulls. Tyrosine phosphorylation of a number of proteins from whole-cell extracts increased in a time-dependent manner during in vitro incubation and was unaffected by the presence of theophylline in the medium. The oxygenation state of the incubation medium had profound effects on sperm motility, viability, and tyrosine phosphorylation of proteins from whole-cell extracts. Sperm motility and viability declined more rapidly under aerobic compared with anaerobic conditions. Tyrosine phosphorylation of proteins from whole-cell extracts increased considerably during anaerobic incubation, while there was no significant change during aerobic incubation. This increase in phosphorylation due to anaerobic incubation was reversed when sperm were transferred from an anaerobic to an aerobic environment, indicating that the oxygenation state of the medium regulates both protein tyrosine kinases and phosphatases. In addition, sperm incubated under aerobic conditions for 5 days retained the ability to phosphorylate proteins when transferred to an anaerobic environment. The increase in protein tyrosine phosphorylation during in vitro incubation took place in a medium that did not contain capacitating substances such as heparin, sodium bicarbonate, or BSA. It transpired over a time scale of days and was not augmented by an increase in intracellular cAMP concentration through phosphodiesterase inhibition. Protein tyrosine phosphorylation during extended in vitro incubation at ambient temperature was significantly inhibited by the presence of oxygen in the medium.