A system for transport of coenzyme M, 2-mercaptoethanesulfonic acid (HS--CoM), in Methanobacterium ruminatium strain M1 required energy, showed saturation kinetics, and concentrated the coenzyme against a gradient. The process was sensitive to temperature and was maximally active at pH 7.1. Cells took up HS--CoM at a linear rate, with a Vmax of 312 pmol/min per mg (dry weight) and an apparent Km of 73 nM. An intracellular pool of up to 5 mM accumulated which was not exchangeable with the medium. Uptake required both hydrogen and carbon dioxide; it was inhibited by O2. Bromoethanesulfonic acid (BrCH2CH2SO3-), a potent inhibitor of methanogenesis in cell-free extracts, inhibited both uptake and methane production. Results of inhibitor studies with derivatives and analogs of the coenzyme showed that the specificity of the carrier is restricted to a limited range of thioether, thioester, and thiocarbonate derivatives. 2-(Methylthio)ethanesulfonic acid (CH3--S--CoM) showed an apparent Ki for HS--CoM uptake of 15 nM, being taken up itself with a Vmax of 320 pmol/min per mg (dry weight) and an apparent Km of 50 nM. An analysis of intracellular pools after HS--CoM uptake indicated that the predominant forms are a heterodisulfide of unknown composition and CH3--S--CoM.