Several laboratories have reported that Na+-Li+ countertransport activities are increased in red blood cells from patients with essential hypertension. It has been proposed that the activity of this red blood cell transport system might reflect the activity of a similar system in vascular smooth muscle. We previously demonstrated Na+-Li+ exchange in sarcolemmal vesicles from canine artery and proposed that this transport function might be mediated by the Na+-H+ exchanger. In the present studies, however, we were unable to demonstrate Na+-Li+ countertransport in canine red blood cells. Since bovine red blood cells have a vigorous Na+-Li+ exchanger and we previously demonstrated Na+-H+ exchange in sarcolemmal vesicles from bovine artery, we wished to determine whether bovine sarcolemmal vesicles mediate Na+-Li+ exchange and whether this transport function is mediated via the Na+-H+ exchanger. We found that an outwardly directed proton or Li+ gradient stimulated 22Na+ uptake in sarcolemmal vesicles from bovine superior mesenteric artery. Li+ gradient-stimulated Na+ uptake was not due to electrical coupling between the two ions, was not affected by a change in membrane potential, and could not be explained by the parallel operation of Li+-H+ and Na+-H+ exchange. External Li+ inhibited proton gradient-stimulated Na+ uptake, and external protons inhibited Li+ gradient-stimulated Na+ uptake. Na+ efflux from vesicles was stimulated by inwardly directed gradients for Li+ or protons, and these effects were not additive. Proton efflux from vesicles was stimulated by inwardly directed gradients for Na+ or Li+, and these effects were not additive. Finally, Na+-H+ exchange and Na+-Li+ exchange in sarcolemmal vesicles were inhibited by 5-(N-ethyl-N-isopropyl)amiloride in an identical dose-dependent manner. In conclusion, Na+-Li+ countertransport could not be demonstrated in canine red blood cells, but as is the case with bovine red blood cells, sarcolemmal vesicles from bovine artery mediate Na+-Li+ countertransport. This transport function and sarcolemmal Na+-H+ exchange are mediated via a single 5-(N-ethyl-N-isopropyl)amiloride-sensitive cation exchanger with affinity for Na+, Li+, and protons. The cow, as opposed to the dog, may be a good animal model to test whether the activity of red blood cell Na+-Li+ countertransport is predictive of the activity of Na+-Li+ (and Na+-H+) exchange in vascular smooth muscle.