Previous results using gene transfection methods have shown that the wild-type (WT) von Hippel-Lindau (VHL) gene can function as a potent tumor suppressor gene in vivo for renal cell carcinoma (RCC) cells in the absence of any suppressive effect on cell growth in monolayer cell culture under serum-rich conditions. Because we had previously found that the function of some oncogenes, such as mutant ras, can be influenced by three-dimensional growth as multicellular spheroids (J. Rak et aL, J. Cell Biol., 131: 1587-1598, 1995), we reasoned the same might be true for suppressor genes as well. We, therefore, decided to compare and study the effects of the WT VHL gene in monolayer versus three-dimensional culture systems of the RCC cell line 786-0, which contains an inactivated VHL gene. We found that the reintroduction of the WT VHL gene into mutant VHL RCC cells resulted in growth suppression in vitro, but only when the cells were grown as spheroid cultures. This decrease in cell proliferation was associated with several features of cell differentiation/morphogenesis, as shown by light and electron microscopy. Thus, in contrast to cultures of mutant VHL RCC cells, which formed very compact and cohesive spheroids, the WT VHL transfectants were loosely arranged and formed a network of tubular and trabecular structures within the spheroids. The morphological changes of the WT VHL spheroids were associated with the deposition of fibronectin in the extracellular space, a feature that was absent in the mutant and inactivated VHL gene-expressing spheroids. The results suggest the VHL gene may be involved in the maintenance of the epithelial phenotype of renal tubular cells, ie., it may act as a differentiation/morphogenetic factor. Moreover, this effect in tumors cells appears to be highly dependent on multicellular growth conditions that mimic the basic nature of solid tumors, such as RCC.