To determine whether glycogen synthase (GS) activity remains impaired in skeletal muscle of non-insulin-dependent diabetes mellitus (NIDDM) patients or can be normalized after prolonged culture, needle biopsies of vastus lateralis were obtained from 8 healthy nondiabetic control (ND) and 11 NIDDM subjects. After 4-6 wk growth and 4 d fusion in media containing normal physiologic concentrations of insulin (22 pM) and glucose (5.5 mM), both basal (5.21 +/- 0.79 vs 9.01 +/- 1.25%, P < 0.05) and acute insulin-stimulated (9.35 +/- 1.81 vs 16.31 +/- 2.39, P < 0.05) GS fractional velocity were reduced in NIDDM compared to ND cells. Determination of GS kinetic constants from muscle cells of NIDDM revealed an increased basal and insulin-stimulated Km(0.1) for UDP-glucose, a decreased insulin-stimulated Vmax(0.1) and an increased insulin-stimulated activation constant (A(0.5)) for glucose-6-phosphate. GS protein expression, determined by Western blotting, was decreased in NIDDM compared to ND cells (1.57 +/- 0.29 vs 3.30 +/- 0.41 arbitrary U/mg protein, P < 0.05). GS mRNA abundance also tended to be lower, but not significantly so (0.168 +/- 0.017 vs 0.243 +/- 0.035 arbitrary U, P = 0.08), in myotubes of NIDDM subjects. These results indicate that skeletal muscle cells of NIDDM subjects grown and fused in normal culture conditions retain defects of basal and insulin-stimulated GS activity that involve altered kinetic behavior and possibly reduced GS protein expression. We conclude that impaired regulation of skeletal muscle GS in NIDDM patients is not completely reversible in normal culture conditions and involves mechanisms that may be genetic in origin.