Studies using adult human islets and mouse embryonic stem cells have suggested that the neurepithelial precursor cell marker nestin also identifies and can be used to purify beta-cell precursors. To determine whether nestin can be used to identify beta-cell progenitors in the developing human pancreas, we characterized nestin expression from 12 to 24 gestational weeks, purified nestin+ cells using an enhancer/promoter-driven selection plasmid, and determined whether nestin+ cells can differentiate into beta-cells. Nestin was visualized in the platelet endothelial cell adhesion molecule and alpha smooth muscle actin-positive blood vessels and colocalized with vimentin in the interstitium. Nestin was not observed in pan cytokeratin (pCK)-positive ductal epithelium or insulin cells. Purified nestin+ cells also coexpressed vimentin and lacked pCK immunoreactivity. Purified adult and fetal pancreatic fibroblasts also expressed nestin. The nestin enhancer/promoter used in the selection plasmid was sufficient to drive reporter gene expression, green fluorescent protein, in human fetal pancreatic tissue. Exposure of selected nestin+ cells to nicotinamide, hepatocyte growth factor/scatter factor, betacellulin, activin A, or exendin-4 failed to induce pancreatic and duodenal homeobox gene-1 or insulin message as determined by RT-PCR. Transplantation of nestin+ cells and fetal pancreatic fibroblasts into athymic mice also failed to result in the development of beta-cells, whereas nestin- fetal pancreatic epithelial cells gave rise to functional insulin-secreting beta-cells. We conclude that nestin is not a specific marker of beta-cell precursors in the developing human pancreas.