Enolase from rabbit muscle (betabeta-enolase) is inactivated by NaClO(4). Enolase free of divalent cations is more susceptible to inactivation by NaClO(4) than is enolase in the presence of Mg(2+). We find that substrate protects apo-enolase against inactivation, indicating that substrate can bind to enolase in the absence of a divalent cation. This binding is not due to contamination by trace levels of divalent cations since (1) it occurs even in the presence of EDTA or EGTA and (2) metal analysis by ICP (inductively coupled plasma) mass spectrometry did not reveal sufficient contamination to account for the protection. The binding of PGA to apo-enolase did require Na(+). When TMAClO(4) was used instead of NaClO(4), there was no protection by PGA. Protection was restored when TMAClO(4) plus NaCl were used. The inactivation of apo-enolase by NaClO(4) is due to dissociation into inactive monomers. We conclude that Na(+) binds to apo-enolase, permitting substrate to then bind. Of the three known Me(2+) binding sites on enolase, we believe the most likely binding site for Na(+) is the carboxylate cluster of site 1, the highest affinity site of enolase.