Two isoforms of creatine kinase (CK, E.C. 2.7.3.2), the brain type BB-CK and the mitochondrial-bound MiMi-CK, as well as adenylate kinase (myokinase, E.C. 2.7.4.3) were identified in washed spermatozoa from chicken and man by cellulose polyacetate electrophoresis and immunoblots. BB-CK was localized by indirect immunofluorescence staining within the sperm tail but not in the head portion. MiMi-CK is confined to the midpiece region rich in mitochondria and has been localized directly by immunogold staining within the mitochondria. In contrast to chicken, seminal plasma from man was also found to contain considerable amounts of BB-CK. Total creatine content of spermatozoa (8-15 mM) and seminal plasma (3.8 +/- 0.4 mM) as well as preliminary experiments with metabolic blockers indicate a dependence of sperm motility on CK and phosphoryl creatine (CP). The presence of two CK isoforms located in different 'compartments' of spermatozoa suggests a CP-shuttle in sperm similar to that described for cross-striated muscle.