Ouabain-resistant Na+-Li+ countertransport was studied on erythrocytes of man, sheep, rabbit, and beef. A transport system, exchanging Li+ for Na+ in a ratio of 1:1, was present in all four species. Li+ uptake by the exchange system increased 30-fold in the order man less than HK-sheep less than LK-sheep less than rabbit less than LK-beef. This order is identical to that of ouabain-resistant Na+-Na+ exchange in these species, but bears no relation to the Na+-K+ pump activity. The activity of the Na+-Li+ exchange system varied up to 7 and 16-fold among individual red cell specimens from man and beef, the variability being much smaller in sheep and rabbit erythrocytes. The affinities of the system for Li+ and Na+ were similar among the species and individuals (half saturation of the external site at about 1 mM Li+ and 50 mM Na+, respectively). 50-60% of Na+-Li+ exchange was blocked by N-ethylmaleimide in all species. p-Chloromercuribenzene sulfonate inhibited the exchange only in beef and sheep erythrocytes (60-80%). The two SH-reagents act by decreasing the maximum activity of the system, whilst leaving its affinity for Li+ unaltered. Phloretin was a potent inhibitor in all species. 1 mM each of furosemide, ethacrynic acid, and quinidine induced only a slight inhibition. The Na+-Li+ exchange of human and beef erythrocytes increased 3.5-fold upon elevation of the extracellular pH from 6 to 8.5, the pH-dependence arising from a change in affinity of the system for the cations and being similar to that reported for ouabain-resistant Na+-Na+ exchange in beef erythrocytes. It is concluded that a transport system exists in the red cell membranes of the four species which can mediate ouabain-resistant exchange of either Na+ for Na+, Na+ for Li+, or Li+ for Li+. The exchange system exhibits essentially identical transport characteristics in the four species, but shows a marked inter- and intra-species variability in maximum transport capacity and some differences in susceptibility towards inhibitors. A similar transport system is probably present also in other tissues. The exchange system seems to be distinct from the conventional Na+-K+ pump and shows no clear relation to one of the furosemide-sensitive, ouabain-resistant Na+ transport systems described in the literature.