Glucokinase (GK) is the rate-limiting enzyme in the glycolytic pathway of the β-cell and, even in the rat fetus at 22-days gestation, immediately before birth, acts as a sensor of glucose influencing the rate of glucose utilization. However, when GK first appears in islets during β-cell development is unknown. Whether GK is expressed in fetal glucagon-producing cells is also unknown. To determine this information, fetal rat islets were examined at 16-, 18-, and 22-days gestation. GK was identified immunocytochemically in bothβ - and α-cells at all these ages, with the number of GK immunoreactive cells positively correlated to the fetal age from 16–22 days. Western blot analysis of islet protein extracts demonstrated the presence of GK, at 52 kDa, at 16 days and thereafter. To determine whether glucose had any effect on regulation of GK biosynthesis, fetal islets were cultured in medium containing a wide range of concentrations of glucose for 7 days. The amount of GK protein was significantly decreased in low concentrations of glucose and augmented at high concentrations. In conclusion, GK was expressed in both β- and α-cells in fetal rat islets during development. GK is an integral part of the function of both of these cells at all stages in the development of the fetal islet.