In sperm of the sea urchin Strongylocentrotus purpuratus, a functional phosphocreatine shuttle, that requires the existence of mitochondrial and cytosolic creatine kinase (CK) isoforms in distinct locations, is essential for sperm motility. S. purpuratus sperm have an unusually large, 145 kDa CK isoform, present exclusively in the sperm tail (TCK), that is enriched in flagellum membrane preparations. Purified TCK contains two very similar proteins, designated TCKI and TCKII, of which only TCKII associates readily with liposomes and detergent micelles in vitro. Here we demonstrate by gas chromatography/mass spectrometry combined with selective ion monitoring that ions diagnostic for the presence of myristoylglycine in proteins are found in TCKII, but not TCKI. By contrast, TCKI, but not TCKII, served in vitro as a substrate for recombinant, polyhistidine-tagged N-myristoyltransferase and was myristoylated to high stoichiometries (0.58 +/- 0.14 pmol of myristate/pmol of TCK), in the presence of myristoyl-CoA, on glycine in amide linkage. In vitro myristoylated TCKI associated with phosphatidylcholine (PC)/phosphatidylserine (PS) (75:25) liposomes and Triton X-100 detergent micelles in gel filtration assays and with PC/PS liposomes in a centrifugation assay in the same manner as did TCKII. In gel filtration experiments, TCKI required at least 25-fold higher PC/PS liposome concentrations than TCKII to obtain 50% association. A partition coefficient of 0.8 x 10(5) M(-1) was determined for TCKII with PC/PS (75:25) liposomes in the centrifugation assay. Thus, myristoylated and nonmyristoylated forms of TCK exist side-by-side in the sea urchin flagellum, and myristoylation is essential for efficient liposome association of TCK.