We have previously shown that when rat jejunum is challenged with high concentrations of glucose, the facilitative transporter GLUT2 is mobilised during Na⁺/coupled transport and inserted into the brush border membrane (Kellett and Helliwell, 2000). GLUT2 then affords the major route of absorption so that absorptive capacity is precisely regulated to match dietary intake. In Caco-2 cells, Na⁺/glucose cotransport induces cytoskeletal rearrangement caused by myosin II regulatory light chain (myosin II RLC₂₀) phosphorylation in the perijunctional actomyosin ring (Turner et al., 1997). We report here that GLUT2 insertion into the brush border membrane of rat jejunum requires myosin II RLC₂₀ phosphorylation. All animal procedures conformed to the UK Animals (Scientific Procedures) Act, 1986. Male Wistar rats were anaesthetised by an I.P. injection of a mixture of 1.0 ml Hypnorm (Janssen Animal Health) and 0.5 ml Hypnovel (Roche Diagnostics) per kilogram body weight. Fed rats were perfused with 75 mM glucose in vivo to insert GLUT2 into the brush border membrane. Myosin light chain kinase (MLCK) phosphorylates myosin II RLC₂₀ inducing actomyosin contraction. ML-7 (5 μM), a cell permeable and potent inhibitor of MLCK inhibited total glucose absorption by ∽ 50 % from a control rate of 38.0 ± 0.5 to 20.2 ± 0.8 μmol min^-1 (g dry weight)^-1 (p 〈 0.001, n = 9). Selective inhibition of the GLUT2 component with phloretin (1 mM) demonstrated that ML-7 inhibited the GLUT2 component from a rate of 20.2 ± 0.8 μmol min^-1 (g dry weight)^-1 in the presence of ML-7 (5 μM) to 10.5 ± 0.6 μmol min^-1 (g dry weight)^-1 in the presence of ML-7 and phloretin (p < 0.001; n = 9). This effect was correlated to a 60.3 ± 1.8 % diminution in apical GLUT2 levels (p < 0.001; n = 3); SGLT1 levels were unaffected. Western blotting of total and phosphorylated myosin II RLC₂₀ showed a 3-fold reduction in this ratio in response to ML-7 and an ∽ 4.5-fold reduction in the absence of glucose. We conclude that initiation of Na⁺/glucose cotransport stimulates Ca⁺⁺ entry through Ca_V1.3 in the brush border membrane (Morgan et al., 2003) resulting in myosin II RLC₂₀ phosphorylation and subsequent actomyosin contraction. The ensuing cytoskeletal rearrangement seems essential to increase the absorptive capacity of the small intestine for glucose via insertion of GLUT2 into the brush border membrane.