Incubation of rabbit skeletal myosin with 1 to 3 mM D-glucose 6-phosphate over a period of several hours resulted in the inhibition of the K(+)- and actin activated-ATPase activities. Substrate ATP (0.5-3 mM final concentration) protected the myosin against the loss of ATPase activity as induced by glucose 6-phosphate. This was also found for ADP. When the myosin was incubated with 3 mM [3H] labeled glucose 6-phosphate for 28 h. up to one mole of glucose 6-phosphate was incorporated per 4.7 x 10(5) g of myosin. A significant reduction in the labeling occurred in the presence of ATP. The labeling was limited to the heavy chain region as judged by gel electrophoresis which resolved the heavy and light chain components of myosin. The non-enzymatic glycation of myosin by glucose 6-phosphate is probably the primary cause for the observed loss of the ATPase activity of myosin. This effect may also occur physiologically modifying the activity of muscle contractile proteins particularly during prolonged hyperglycemia.