A recent report provides new evidence for the presence of glucokinase (GK) in the anterior pituitary. In the present study, immunohistochemistry was used to identify the cells containing GK in the pituitary of rats and monkeys. In rats, GK was detected as a generalized cytoplasmic staining in a discrete population of cells in the anterior pituitary. In colocalization experiments, the majority of cells expressing follicle-stimulating hormone (FSH) or luteinizing hormone (LH) also contained GK. In addition to the gonadotropes, GK was observed in a subpopulation of corticotropes and thyrotropes. GK was not detected in cells expressing growth hormone or prolactin. In monkeys, GK was also observed in a discrete population of cells. Intracellular distribution differed from the rat in that GK in most cells was concentrated in a perinuclear location that appeared to be associated with the Golgi apparatus. However, similar to rats, colocalization experiments showed that the majority of cells expressing FSH or LH also contained GK. In addition to the gonadotropes, GK was observed in a subpopulation of corticotropes and thyrotropes. In the monkey, only a few cells had generalized cytoplasmic staining for GK. These experiments provide further evidence for the presence of GK in the anterior pituitary. Although some corticotropes and thyrotropes contained GK, the predominant cell type expressing GK was gonadotropes. In view of the generally accepted role of GK as a glucose sensor in a variety of cells including the insulin-producing pancreatic β-cells as the prototypical example, it is hypothesized that hormone synthesis and/or release in pituitary cells containing GK may be directly influenced by blood glucose.