ATP level, cell motility and viability, oxygen uptake, pyruvate kinase activity, and ultra-weak photon emission (UPE) induced by red-ox Fe(2+)-ascorbate cycling system were studied in fresh, in previously equilibrated in a glycerol diluent, and in cryopreserved bull spermatozoa, exposed to thermal stress by incubation of the cells at 44 degrees C. A sharp drop in motility and viability of fresh spermatozoa and even more so, of equilibrated and cryopreserved cells was accompanied by accumulation of ATP. When cell movement was totally inhibited, ATP utilization was decreased, while chemical energy continued to be produced by cell pyruvate kinase, one of the key glycolytic enzymes, which in spermatozoa is very active (6500 IU/g protein) and insensitive to feed-back inhibition by excess of ATP and L-cysteine. Accumulation of ATP during incubation at 44 degrees C in 0.9% NaCl was accompanied by rapid decrease in oxygen consumption by fresh spermatozoa and an increase in Fe(2+)-ascorbate induced UPE, followed by a sharp decrease in ATP level observed at the end of induced UPE measurement. The increase in photon emission due to lipid peroxidation was highly correlated with the increase in cell ATP level caused by thermal stress.