Patients with non-insulin-dependent diabetes mellitus (NIDDM) exhibit insulin resistance and decreased glucose transport in skeletal muscle. Total content of muscle GLUT4 protein is not affected by NIDDM, whereas GLUT4 mRNA content is reported, variously, to be unaffected or increased. Physical training is recommended in the treatment of NIDDM, but the effect of training on muscle GLUT4 protein and mRNA content is unknown. To clarify the effect of training in NIDDM, seven men with NIDDM (58 ± 2 years of age [mean ± SE]) and eight healthy men (59 ± 1 years of age) (control group) performed one-legged ergometer bicycle training for 9 weeks, 6 days/week, 30 min/day. Biopsies were obtained from the vastus lateralis leg muscle before and after training. GLUT4 protein analyses was performed along with analyses of muscle biopsies from five young (23 ± 1 years of age) (young group), healthy subjects who participated in a previously published identical study. In response to training, maximal oxygen uptake increased (Δ 3.3 ± 1.8 in NIDDM subjects and 4.5 ± 1.2 ml.min−1·kg−1 in control subjects [both P < 0.05]). Before training, GLUT4 protein content was similar in NIDDM, control, and young subjects (0.35 ± 0.02, 0.34 ± 0.03, and 0.41 ± 0.03 arbitrary units, respectively), and it increased (P < 0.05) in all groups during training (to 0.43 ± 0.03, 0.40 ± 0.03, and 0.57 ± 0.08 arbitrary units, respectively). GLUT4 mRNA content was always lower in NIDDM compared with control subjects (P < 0.05) and increased in both groups (P < 0.05) during training (94 ± 6 to 122 ± 8 and 151 ± 5 to 170 ± 4 arbitrary units/10 μg total RNA, respectively). We conclude that muscle GLUT4 protein and mRNA increase in both NIDDM and control subjects in response to training. GLUT4 mRNA content is lower in NIDDM subjects compared with control subjects. GLUT4 protein content does not change with age.