Our objective was to determine if cultured strains of rumen bacteria would transport a fluorescent analog of glucose (2-NBDG) with the same specificity and kinetics as glucose. Our rationale was that 2-NBDG could be used to identify uncultured, glucose-utilizing bacteria if it were transported similarly to glucose. Pure cultures of bacteria were harvested in the mid-to-late log phase, washed, and dosed with 2-NBDG or radiolabeled sugar (0 to 100 µM). Transport was halted by adding −5°C stop buffer and filtering through a membrane. The membrane was taken for fluorometry or liquid scintillation counting. Transport of 2-NBDG could be detected within 2 s of 2-NBDG addition for Streptococcus bovis and Selenomonas ruminantium (2 strains each), but it was not detected at any time for 6 other glucose-fermenting species. Genomes of S. bovis and S. ruminantium strains were found to possess genes for the mannose phosphotransferase system, whereas the other species had genes for other glucose transporters. For S. bovis JB1, the Michaelis constant (K_m) for 2-NBDG transport was 10.6-fold lower than that for [¹⁴C]-glucose transport (P = 0.006). The maximum velocity (V_max) was 2.9-fold lower than that for [¹⁴C]-glucose, but this difference was not significant (P = 0.100). In another set of experiments, transport of 2-NBDG at a single concentration (100 μM) was compared with that of [¹⁴C]-glucose, [³H]-mannose, and [¹⁴C]-deoxy-2-glucose. For S. bovis JB1, transport of 2-NBDG was 3.2-fold lower than that of [¹⁴C]-glucose (P = 0.002) but similar to that for [³H]-mannose (P = 0.992) and [¹⁴C]-deoxyglucose (P = 0.955). 2-NBDG could identify uncultured, glucose-utilizing bacteria, but only those with a mannose phosphotransferase system (not other glucose transporters). Its transport may more closely reflect that of mannose and deoxy-2-glucose than glucose.