One of the major β-cell autoantigens associated with IDDM is GAD. Although GAD expression has been detected in adult islets, transcriptional expression of the GAD genes has not been reported during human pancreatic ontogeny. We therefore analyzed patterns of GAD gene transcription by quantitating the mRNAs encoding both the 65- and 67-kDa isoforms (GAD₆₅ and GAD₆₇, respectively) in human fetal, postnatal, and adult pancreases, as well as in isolated adult islets, and examined their tissue-specific expression. Significant levels of pancreatic GAD₆₅ transcripts were already detected at 13 weeks of gestation and were expressed at higher levels in the fetal and infantile pancreas than in the adult pancreas. Isolated adult pancreatic islets were highly enriched in GAD₆₅ mRNA. In contrast, GAD₆₇ transcripts were not detectable in fetal and postnatal pancreases. In addition to the pancreas, marked GAD expression was detected in the brain, whereas other tissues examined contained either low or undetectable GAD transcripts. Triple immunofluorescent staining of fetal and adult pancreases revealed colocalization of GAD₆₅ with α- and β-cells. In the fetal pancreas, strong immunoreactivity for GAD₆₅ was also evident in epithelial cells, which lacked expression of insulin or glucagon, some of which were present in the ductal epithelium, suggesting that GAD₆₅ expression might correlate with endocrine determination. In summary, 1) this is the first demonstration of GAD₆₅ expression in the human fetal pancreas, implicating a potential role during islet development, and 2) GAD₆₅ may be a useful marker for the identification of primitive islet cells.