When sucrose is present in the external medium, it is internalized by Methanococcus thermolithotrophicus. Sucrose internalization, as determined by both natural abundance C-13 nuclear magnetic resonance spectroscopy and [C-14]sucrose uptake, is directly proportional to external sucrose levels. The uptake is energy independent and exhibits kinetic behavior consistent with a simple passive diffusion process. In the presence of 0.2 M sucrose, methanogenesis is inhibited as the NaCl concentration in the external medium is increased. Growth, as determined by protein content, is inhibited by 0.2 M sucrose when the external NaCl concentration is 1.4 M. These results are important,because they show that (i) sucrose cannot be used as a noncharged solute to replace NaCl in experiments to evaluate how external osmotic strength affects the internal solute composition of M. thermolithotrophicus, and (ii) sucrose cannot be used as an impermeable marker for the extracellular volume in experiments to measure the intracellular volume of M. thermolithotrophicus.