Pancreatic intraepithelial neoplasia (PanIN) is a precursor to invasive ductal adenocarcinoma of the pancreas. Observations made in genetically engineered mouse models suggest that the acinar/centroacinar compartment can give rise to ductal neoplasia. To integrate findings in mice and men, we examined human acinar cells, acinar-ductal metaplasia (ADM) lesions, and PanINs for KRAS2 gene mutations. Surgically resected pancreata were screened for foci of ADM with or without an associated PanIN lesion. Stromal cells, acinar cells, ADMs, and PanINs were separately isolated using laser capture microdissection. KRAS2 status was analyzed using genomic DNA isolated from the microdissected tissue. Twelve of these 31 foci of ADM occurred in isolation, whereas 19 were in the same lobules as a PanIN lesion. All 31 microdissected foci of acinar cells were KRAS2 gene wild-type, as were all 12 isolated ADM lesions lacking an associated PanIN. KRAS2 gene mutations were present in 14 of 19 (74%) PanIN lesions and in 12 of the 19 (63%) foci of ADM associated with these PanINs. All ADM lesions with a KRAS2 gene mutation harbored the identical KRAS2 gene mutation found in their associated PanIN lesions. Ductal neoplasms of the human pancreas, as defined by KRAS2 gene mutations, do not appear to arise from acinar cells. Isolated AMD lesions are genetically distinct from those associated with PanINs, and the latter may represent retrograde extension of the neoplastic PanIN cells or less likely are precursors to PanIN.