In the spermatozoa of some species creatine kinase (CK: E.C. 2.7.3.2) is involved in shuttling energy, in the form of creatine phosphate, between the mid-piece mitochondria and flagellum. In this study, the effects of the CK inhibitor dinitrofluorobenzene (DNFB) on human sperm CK activity, motility and ATP concentrations were assessed with different energy substrates. There was a dose-dependent inhibition of CK activity by DNFB but inhibition was incomplete and there was no effect on the percentage of flagellating cells, irrespective of substrate. However, when lactate alone supported the cells DNFB decreased velocities and increased amplitude of head displacement (fewer progressively motile forms were observed), whereas ATP concentrations in spermatozoa were unaltered. Demembranated sperm models could be reactivated by ADP plus creatine phosphate, but not to the extent caused by ATP, and were able to be inhibited by myokinase inhibitors. Increased velocities, linearity (LIN) and beat cross frequency (BCF) were demonstrated for spermatozoa incubated with lactate, in contrast to glucose as sole energy source, and higher velocities and BCF were generated in the presence of both substrates. This suggests that the production of ATP by glycolysis and respiration are independent and complementary. CK is not obligatory for sperm motility but supplements energy provision under certain conditions.