Limulus spermatozoa are nonmotile after dilution into sea water, and motility is initiated in situ by a sperm motility-initiating peptide (SMI) that emanates from Limulus eggs. In the absence of SMI, several structurally different chelators (EDTA, EGTA, phenanthroline, dipyridyl, cysteine, and dithiothreitol; 1 mM each) initiated sperm motility; and in the presence of SMI, EDTA prolonged the duration of motility initiated by SMI. Of the elements determined to be present in Limulus sperm, Zn+2 (0.1 pM free Znf2) was both the most effective at preventing EDTA initiation of motility and the only ion to prevent EDTA prolongation of SMI-initiated motility. 65Zn was concentrated from sea water by Limulus sperm and was rapidly removed by EDTA. These results indicate that removal of the Zn+2 normally associated with Limulus spermatozoa can initiate or enhance motility. However, SMI does not act as a Zn+2 chelator, since SMI removed little 65Zn from the sperm while at the same time initiating motility of greater percentage and duration than did EDTA. The mechanism by which Znf2 removal enhances sperm motility is not yet known but could involve a Znf2-dependent or Znf2-regulated step in the pathway of SMI action.