The main characteristic of polycystic kidney disease is the development of multiple fluid filled renal cysts. The discovery of mislocated Na,K-ATPase in the apical membrane of cyst lining epithelia alluded to a reversal of polarity as a possible explanation to the fluid secretion. The topic of apical Na,K-ATPase in cysts remains controversial. We investigated the localization of the Na,K-ATPase and assessed the apical-basolateral polarization of cyst lining epithelia by means of immunohistochemistry in kidney tissue from six polycystic kidney disease patients undergoing nephrectomy. The Na,K-ATPase α1 subunit was conventionally situated in the basolateral membrane of all immunoreactive cysts. Proteins of the Crumbs and partitioning defective (Par) complexes were localized to the apical membrane domain in cyst epithelial cells. The apical t-SNARE protein Syntaxin-3 also immunolocalized to the apical domain of cyst lining epithelial cells. Proteins of the basolateral Scribble complex immunolocalized to the basolateral domain of cysts. Furthermore, we confirmed that cysts can originate from virtually any tubular segment with preserved polarity. In conclusion, we find no evidence of a reversal in apical-basolateral polarity in cyst lining epithelia in polycystic kidney disease.