Clostridium pasteurianum is able to build up about 100-fold gradients of methylammonium across the cell membrane. Methylammonium enters the cell by means of a carrier as shown by the energy requirement, saturation kinetics and a pH profile with a narrow maximum between pH 6.2 and 6.8. The methyl ammonium transport (apparent Km = 150 microM, V = 100 mumol/min per g dry weight) is competitively inhibited by ammonium (apparent Ki = 9 microM). The low Ki value and the observation that methylammonium cannot serve as a carbon or nitrogen source for Cl. pasteurianum strongly indicate that ammonium rather than methylammonium is the natural substrate. Uncouplers and inhibitors of energy metabolism or of the membrane-bound ATPase inhibit transport. Cl. pasteurianum maintains a membrane potential (interior negative) in the range 80-130 mV. This membrane potential was identified as the energy source: the same agents that block transport also decrease the membrane potential, and artificial generation of a membrane potential (by addition of valinomycin to K+-loaded cells) induces concentrative uptake of methylammonium. Thus NH4+ (or CH3NH3+) must be the transported species. Digestion of the cell wall by lysozyme does not abolish the transport activity.