Red blood cells usually replenish their ATP pools by glycolysis, fueled by glucose imported via the cell membrane. Mature red cells of some species (e.g. pig, chicken) have, however, been reported to show very low glucose transport. The subject of this study was the possible dependency of the level of a key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAD) on glucose transporter activity during the maturation of chicken red cells. The chicken pronormoblast cell line, HD3, was used as a model system. These cells contain higher levels of GAD and glucose transporter activities than normal chicken bone marrow cells, but reduce their levels during maturation. In an attempt to assess whether the decrease in GAD activity is regulated by the glucose transport, the chicken GLUT3 expressed under the control of viral promoter was introduced into HD3 cells by retroviral infection (pDOL-cGT3). Upon cell differentiation and maturation, both cells with and without the exogenous transporter decreased GAD activity. Butyric acid did not affect the regulation of GAD activity upon differentiation. These results show that the development of chicken red cells is manifested by reduction of their GAD activity and that this is not affected by their sugar transporter activity. The very low GAD activity in embryonic chicken red cells is thus due to a loss of this activity at an early stage in their development. Because of the very low glucose transport and GAD activities in mature chicken red cells, rates of glycolysis are likely to be low and suggesting an alternative pathway for ATP production in these cells.