The kidney is affected by many chemicals. Some of the chemicals may even contribute to end-stage renal disease and thus contribute considerably to health care costs. Because of the large functional reserve of the kidney, which masks signs of dysfunction, early diagnosis of renal disease is often difficult. Although numerous studies aimed at understanding the mechanisms underlying chemicals and drugs that target various renal cell types have delivered enough understanding for a reasonable risk assessment, there is still an urgent need to better understand the mechanisms leading to renal cell injury and organ dysfunction. The increasing use of in vitro techniques using isolated renal cells, nephron fragments, or cell cultures derived from specific renal cell types has improved our insight into the molecular mechanisms involved in nephrotoxicity. A short overview is given on the various in vitro systems currently used to clarify mechanistic aspects leading to sublethal or lethal injury of the functionally most important nephron epithelial cells derived from various species. Whereas freshly isolated cells and nephron fragments appear to represent a sufficient basis to study acute effects (hours) of nephrotoxins, e.g., on cell metabolism, primary cultures of these cells are more appropriate to study long-term effects. In contrast to isolated cells and fragments, however, primary cultures tend to first lose several of their in vivo metabolic properties during culture, and second to have only a limited life span (days to weeks). Moreover, establishing such primary cultures is a time-consuming and laborious procedure. For that reason many studies have been carried out on renal cell lines, which are easy to cultivate in large quantities and which have an unlimited life span. Unfortunately, none of the lines display a state of differentiation comparable to that of freshly isolated cells or their primary cultures. Most often they lack expression of key functions (e.g., gluconeogenesis or organic anion transport) of their in vivo correspondents. Therefore, the use of cell lines for assessment of nephrotoxic mechanisms will be limited to those functions the lines express. Upcoming molecular biology approaches such as the transduction of immortalizing genes into primary cultures and the utilization of cells from transgenic animals may in the near future result in the availability of highly differentiated renal cells with markedly extended life spans and near in vivo characteristics that may facilitate the use of renal cell culture for routine screening of nephrotoxins.