Three further tests of the Na +-gradient hypothesis are applied. These, like one used earlier, support the hypothesis, which is therefore considered to be established. The findings with the three new tests are as follows. (1) Two Na ions enter the cells in concert with one glycine, as expected from the previously reported kinetic dependence of glycine entry on (Na4)2. (2) A system with high but equal concentrations of Na+ inside and out, which does not pump glycine due to the absence of a Na+ gradient, can be caused to pump glycine (out) by a Donnan effect. In the presence of the Donnan electrical potential there is a Na +-electrochemical gradient even though there is no Na+-concentration gradient. (3) No correlation is found between the con-* The work described in this paper was supported by research grants to Professor F. Haurowitz from the National Science Foundation (NSF G16345