Forskolin (FSK) is an activator of the cAMP signalling cascade that increases D-glucose (D-Glc) transport across the brush-border membrane of the enterocyte (Moreto et al. 1984; Sharp & Debnam, 1994). We have now studied the cellular mechanisms involved in FSK-stimulation of apical D-Glc uptake. The experiments were carried out using 4- to 6-week-old chickens. The everted jejunum was incubated for 20 min at 25°C in a medium with or without FSK (50 μM), and in the presence of the trafficking inhibitors wortmannin (80 μM; WORT) and brefeldin A (0.25 mM; BFA), or the combination of FSK plus WORT or FSK plus BFA. In parallel experiments, the jejunum was also incubated with a combination of FSK plus the unspecific protein kinase inhibitor staurosporine (1 μM; STA) or the protein kinase A inhibitor H8 (60 μM). After incubation, brush-border membrane vesicles (BBMV) were prepared from mucosal scrapings and used for determination of the kinetic constants of D-Glc uptake, and specific phlorizin and cytochalasin B binding to BBMV, as described previously (Garriga et al. 1999). Experiments were approved by the Ethical Committee of the University of Barcelona. Table 1 shows that FSK strongly stimulated maximal apical D-Glc transport rate (V_max). In contrast, incubation with WORT and BFA reduced D-Glc V_max by 30% and 25%, respectively. When vesicles were incubated with FSK together with WORT or BFA, no stimulation of D-Glc V_max was observed. Addition of inhibitors of vesicle trafficking prevented FSK-induced D-Glc V_max stimulation, and the same results were found using protein kinase inhibitors. No effects were observed on Michaelis or diffusion constants in any experimental condition. The absence of specific cytochalasin B binding to BBMV indicated that no GLUT2 was present in BBMV. Specific phlorizin binding measurements demonstrated that the changes in D-Glc V_max were due to the changes in the number of SGLT1 present in BBMV. We conclude that in the chicken jejunum stimulation of D-Glc uptake by FSK is mediated by changes in SGLT1 trafficking from cytoplasmatic compartments to the brush-border membrane. Results also indicate that activation of protein kinase A is involved in this regulatory mechanism.