The glucose transport of mixed ruminal bacteria harvested from a holstein cow fed 5.0 kg of Italian ryegrass and 1.5 kg of flaked corn a day was investigated, The Eadie-Hofstee plot characterized two transport systems: a high-affinity, low-velocity system and a low-affinity, high-velocity system, The former system (K-m = 16 mu M; V-max = 2.2 nmol/min/mg of protein) is considered dominant under this feeding condition based on the glucose concentration in the rumen (<1 mM). In light of the facts that the protonophore SF6847 and the lipophilic triphenylmethyl phosphonium ion had no effect on the high-affinity system and an artificially generated proton gradient and electrical potential across the cell membrane did not increase glucose transport, a proton motive force is not be involved in the system, On the other hand, from the facts that chlorhexidine inhibited about 90% of the high-affinity system while iodoacetate showed no significant effect, and a high phosphoenolpyruvate-dependent phosphorylation of glucose was actually shown, the phosphoenolpyruvate-dependent phosphotransferase system is considered the main system in the high-affinity system, Moreover, as shown by the facts that harmaline inhibited about 30% Of the high-affinity system and the artificially generated sodium gradient across the cell membrane significantly stimulated glucose transport, this system also includes sodium symport to some degree, The high-affinity system was sensitive to a decrease in pH (<6.5) and was inhibited by the presence of sucrose, mannose, and fructose.