Thimerosal activates the production of endothelium-derived relaxing factor (EDRF). I examined whether thimerosal also causes the release of an endothelium-dependent hyperpolarizing factor (EDHF). Thimerosal caused an endothelium-dependent hyperpolarization of smooth muscle. This effect is unlikely to be caused by the property of thimerosal to inhibit the acyl-coenzyme A: lysolecithin acyltransferase (LAT) since neither arachidonic acid nor lysolecithin hyperpolarized the muscles. I conclude that thimerosal is not a pure activator of EDRF (nitric oxide) production, but that it also releases a distinct EDHF.