The Golgi apparatus is a key element in the ordered movement of secretory polypeptides from the rough endoplasmic reticulum to the plasma membrane during secretion. It has been shown that cisternae that receive membranes from the reticulum are morphologically similar to the latter and that cisternae liberating secretory granules resemble that plasma membrane. By using an ultrastructural probe for membrane cholesterol, filipin, on freeze-fractured and thin-sectioned exocrine and endocrine pancreatic cells, we have shown that an enrichment in filipin-cholesterol complexes takes places across the stacked cisternae of the Golgi apparatus; the reticulum-related (forming) cisternae are poor in such complexes, but the secretory granule-related (maturing) cisternae contain numerous complexes. Secretory granule membrane is also richly labeled with filipin-cholesterol complexes. The heterogeneous cholesterol distribution in the membranes of the Golgi apparatus, as shown by filipin, emphasizes the polarity of this organelle, in agreement with its role in organizing the traffic of the secretory polypeptides from the rough endoplasmic reticulum to the plasma membrane.