The established renal epithelial cell line LLC-PK₁ retained in tissue culture several differentiated properties of renal proximal tubular cells. By adapting LLC-PK₁ cells to glucose-free culture conditions, we recently succeeded in isolating a gluconeogenic strain of LLC-PK₁ cells capable of growing in the absence of hexoses. In contrast to the parental wild type, the isolated strain expressed fructose-1,6-bisphosphatase activity and was, therefore, designated LLC-PK₁-FBPase⁺. Besides the differences in glucose metabolism, the isolated gluconeogenic substrain differs form the parental wild type with respect to morphological appearance and the expression of apical membrane marker enzymes. LLC-PK₁-FBPase⁺ cells display a drastic accumulation of autophagic vacuoles, disappearance of apical membrane alkaline phosphatase activity, and increased γ-glutamyltranspeptidase activity. In order to find out whether or not a low alkaline phosphatase activity in combination with the enhanced formation of autophagic vacuoles is related to a change in apical membrane surface, we utilized a combined light and electron microscopic morphometric procedure to determine the absolute amount of organelle volumes and membrane surface areas. This stereologic approach shows that LLC-PK₁-FBPase⁺ cells display a tenfold increase in the volume of autophagic vacuoles and the lysosomal compartment. Analysis of lysosomal enzyme activities, however, revealed no changes as compared to wild-type cells. The apical membrane surface of gluconeogenic cells was found to be increased by 80%. Karyotype analysis revealed that LLC-PK₁ wild-type cells were diploid, whereas FBPase⁺ cells exhibited polyploidy with a high percentage of tetraploid nuclei. Culturing LLC-PK₁-FBPase⁺ cells in the presence of 5 mM glucose does not abolish the morphological and biochemical changes described, indicating the stability of the FBPase⁺ strain.