Two different methods have been used to determine the interfacial potential at the disk membranes of intact isolated bovine rod outer segments: (1) The photolysis products of rhodopsin are known to be dependent on pH. We have used this property in order to probe the interfacial potential at disk membranes which is considered to change the surface pH at the disk membrane seen by rhodopsin. (2) The pK value of the amphiphilic pH-indicating dye neutral red (uncharged basic form) in water is 6.6, but adsorbed to disk membranes at least 7.8. This makes the distribution of neutral red between disk membranes and bulk water dependent on the interfacial potential at the disk membrane if the pH in the bulk solution is less than 7.8. Both methods yielded comparable results on the influence of ions and ion carriers on the interfacial potential at disk membranes. In particular, we have studied the effect of different occupation states on the internal binding capacity (of rod outer segments) for divalent cations. In the presence of the ionophore A23187, addition of EDTA to a suspension of intact rod outer segments removed all endogenous divalent cations (Schnetkamp, P.P.M. (1979) Biochim. Biophys. Acta 554, 441--459) and resulted in an interfacial pH at the disk membrane surface of about 6.4, whereas the bulk pH was 7.4. Subsequent addition of 2 mM Mn2+ saturated the internal binding capacity and resulted in an apparent shift towards alkaline pH of the surface pH at the disk membrane by 1.0--1.1 pH units. This could indicate a change of the interfacial potential by 60--65 mV. The same change of ionic conditions resulted in a change of the interfacial potential by 72 mV as determined from the partitioning behaviour of neutral red. These results were independent of the presence of H+ ionophores such as carbonyl cyanide p-trifluoromethoxy-phenylhydrazone and gramicidin. We conclude that the above results can be explained by the presence of fixed net negative charges (charge density: 0.5--1.5 electronic charges/rhodopsin molecule) at the intradiskal membrane surface. That the above charge density can be attributed to the intradiskal membrane surface is inferred from the observation that the presence of A23187 was required for access of divalent cations to the membrane interface involved in both rod outer segments with an intact as well as with a leaky plasma membrane.