To study the structural features of fetal rat duodenal mucosa associated with histogenesis of villi, duodena from 15- to 19-day fetuses were examined by light and electron microscopy. The duodenal epithelium of 15- to 18-day fetuses was stratified. Distinctive junctional complexes associated with membrane-bounded vesicles and cilia-like structures were seen in the deeper epithelial layers at 15 and 16 days. Small lumina, designated "secondary lumina," lined with a variable number of microvilli developed between epithelial cells at these junctional complexes during the sixteenth through eighteenth days. Degenerative changes and exfoliation of superficial epithelial cells were obvious in 17- and 18-day fetuses. In 18-day fetuses, aggregates of mesenchyme had invaginated the basal aspect of the stratified epithelium. Concomitantly, the number of epithelial layers overlying these mesenchymal projections was decreased. In 19-day fetuses, well formed, short duodenal villi lined by a simple columnar epithelium which included goblet and endocrine cells were evident. Injection of ferritin into the main duodenal lumen of 17-day fetuses failed to reveal continuity between the main lumen and the secondary lumina. However, continuity between many secondary lumina and the main lumen was demonstrated in 18-day fetuses. Thus, major morphological features associated with villus formation in fetal rat duodenum include: (1) formation of many secondary lumina in primitive stratified epithelium, (2) eventual fusion of these lumina with the main duodenal lumen, by their continued growth coupled with exfoliation of degenerating superficial layers and (3) upward growth of mesenchyme towards the lumen as cell exfoliation and expansion of secondary lumina take place.