The glucose-6-phosphatase (G6Pase) system participates in the regulation of glucose homeostasis by converting glucose-6-phosphate (G6P) into glucose and inorganic phosphates. We have used an RT-PCR-based cloning and sequencing approach to study the expression of components of the G6Pase system in the hypothalamus and cortex tissues of the ob/ob mouse. We observed the expression of hepatic G6Pase catalytic subunit, G6PC, in both tissues, although increased template inputs were required for its detection. Conversely, expression of both the mouse homologue of the previously-described brain-specific G6P translocase T1 (G6PT1) variant and of the hepatic G6PT1 isoform was easily detectable in hypothalamus and cortex tissues. Of the proposed G6Pase catalytic subunit homologues, the expression of murine ubiquitous G6Pase catalytic subunit-related protein (UGRP, G6PC3) was also easily detectable in both tissues. However, islet-specific G6Pase catalytic subunit-related protein (IGRP, G6PC2) was expressed in a tissue-specific manner, and was detectable only in hypothalamus tissue at increased template inputs. We conclude that cells within ob/ob mouse hypothalamus and cortex tissues express genes with either established or proposed roles in G6P hydrolysis.