This study examined changes in skeletal muscle GLUT4 content and glucose transport in isolated muscle membranes (GT) from horses before and 2 min after standardised submaximal exercise tests (SET) prior to and after completion of 6 weeks of training. Seven horses, age 3-9 years, body mass mean +/- s.e. 530 +/- 19 kg, and sedentary for at least 4 months, completed 6 weeks of training on a treadmill. An initial SET (UT) was performed on a 4 degree incline at a speed equivalent to 55% of pretraining VO2max and was repeated post-training at the same absolute workload (ABS). A third SET (REL) was performed at 55% of post-training VO2max. There was no significant pre- to postexercise change in GLUT4 content before or after training. Following training, total GLUT4 content was increased 2- or 3-fold in pre-exercise biopsies (pre UT: 0.30 +/- 0.05; pre ABS: 1.05 +/- 0.32; pre REL: 1.34 +/- 0.28 arbitrary units) (P<0.05) with similar increases in postexercise GLUT4 content (P<0.05) (post UT: 033 +/- 0.06; post ABS: 1.19 +/- 0.44; post REL: 1.43 +/- 0.31). GT increased 2.5- to 6-fold in postexercise muscle membrane vesicles in UT over a range of glucose concentrations. After training (ABS and REL), there was a 25-50% attenuation (P<0.05) in membrane GT in response to exercise in ABS and REL. These finding indicate that moderate intensity exercise training increased middle gluteal muscle GLUT4 content, but this change was not reflected in an increase in muscle membrane glucose transport activity in postexercise muscle samples.