The intestinal Na-dependent d-glucose co-transporter (SGLT)-1 in sheep is under dietary regulation by luminal substrates. The aim of the present study was to find out whether the SGLT-1 in the forestomach of sheep is also regulated by sugars. Furthermore, the location of a possible glucosensor (luminal v. intracellular v. basolateral) was to be elucidated. Ruminal epithelia of sheep (Ovis aries, Merino breed) were pre-incubated in Ussing chambers with various substrates on the mucosal (i.e. luminal) or serosal (i.e. blood) side. This pre-incubation period was followed by a second pre-incubation period without the tested substrates (washout period). Thereafter, apical d-glucose uptake by ruminal epithelial cells was determined with 200 mumol d-[C-14]glucose/l in the absence or co-presence of the SGLT-1 inhibitor, phlorizin. Pre-incubation with d-glucose on the mucosal side had no significant effect on apical d-glucose uptake (P>0.05). In contrast, pre-incubation with d-glucose, d-mannose, 3-O-methyl-d-glucose or sucrose on the serosal side significantly increased d-glucose uptake compared with mannitol-treated controls (P<0.05). Serosal pre-incubation with cellobiose or d-xylose had no effect. The stimulation of d-glucose uptake by serosal d-glucose pre-incubation was concentration dependent, with maximal stimulation at about 10 mmol/l. We conclude that the ruminal SGLT-1 can be up-regulated in a concentration-dependent manner by blood-borne d-glucose via an extracellular sugar-sensing mechanism.