Endogenous inhibitors of Na,K-ATPase and ouabain-binding were partially purified from bovine central nervous system, and some of their properties were studied. They were eluted as low-molecular-weight fractions by gel filtration. They could be adsorbed by both Amberlite IR 120 and Amberlite IRA 400 at acidic and basic pH, respectively, indicating that they could act as both anions and cations at different pH. These inhibitors of ouabain-binding appeared to affect specific binding of ouabin, and Scatchard plot analysis showed that the inhibition was competitive, suggesting that they could bind to the same site as ouabain, presumably to Na,K-ATPase itself. The inhibitory activities were heat stable, but charring inactivated them completely.