Gram-negative, ruminal Prevotella strains (n = 15) differed greatly in their sensitivity to the feed additive monensin. Strains that were repeatedly transferred with sublethal doses tolerated more monensin than those that were unadapted, but growth experiments indicated that the sensitivity range was as great as 2000-fold. Prevotella bryantii B(1)4 grew with monensin concentrations as high as 20 mu M, but P. ruminicola H15a, D31d, 20-63, E40a, and D42f never initiated growth if monensin was greater than 0.01 mu M. Washed cell, preparations that were energized with glucose lost intracellular potassium when monensin was added, and potassium depletion could also be used as an index of monensin sensitivity. Adapted cells of P. bryantii B(1)4 had a half-maximal potassium depletion constant (K-d) of 3.2 mu M, but the Kd values of P. ruminicola strains H15a, D31d, 20-63, E40a, and D42f were less than 0.04 mu M Maximal potassium depletion (K-max) Values range from 90% to 40%, and monensin-adapted cells always had lower K-max values than unadapted cells. A linear regression of log K-d/K-max versus percentage decrease in optical density divided by monensin concentration had an r(2) of 0.75, and this regression indicated that potassium depletion from washed cells closely correlated with growth inhibition. P. bryantii B(1)4 had a K-d/K-max ratio that was sevenfold greater than other Prevotella strains, and this result indicated that P. bryantii may be unusual in its ability to grow with very high concentrations of monensin.