The effect of changes in carbohydrate status on the synthesis of specific proteins was investigated in millet (Pennisetum americanum L., Leeke, Tift 23B(1)E(1)) seedlings grown in sterile solution culture. Carbohydrate status was altered by extended darkness and sucrose feeding. Root proteins from intact seedlings were labeled with [(35)S]methionine, phenol-extracted, separated by two-dimensional gel electrophoresis, and visualized by autoradiography. In four separate experiments, two proteins showed a consistent change in labeling when root carbohydrate levels were varied between 200 and 1000 micromole hexose per gram residual dry weight. Labeling of the first protein (P(47), M(r) 47 kD) increased as the carbohydrate levels rose above 500 micromole hexose per gram residual dry weight. Labeling of the second protein (P(34), M(r) 34 kD) increased as carbohydrate levels declined from 500 to 200 micromole hexose per gram residual dry weight. Under extreme conditions, when carbohydrate levels fell below 100 micromole hexose per gram residual dry weight, the labeling pattern of most proteins was drastically altered. It is suggested that P(47) and P(34) are ;carbohydrate responsive proteins,' i.e. proteins whose concentrations are controlled either directly or indirectly by tissue carbohydrate status. In contrast, the changes in protein labeling that occur once carbohydrate pools are depleted may be involved in adaptation to periods of prolonged starvation.