Urea cycle is a hepatic metabolic pathway involving five enzymes and several intermediary metabolites and can be altered by different chemicals. To investigate the effect of arsenic, an ubiquitous hepatotoxic agent, on urea production we exposed long-term cultures of adult rat hepatocytes, which produce urea, to 1.33 and 6.67 pM arsenite for 2 weeks. In cultures exposed to 6.67 pM, urea production decreased 60-70% and cellular arginase activity decreased 30, 70 and 85% after 4, 7 and 14 days of exposure, respectively. The arginase activity released to the medium increased significantly after 4, 7 and 14 days, with a maximum value after 7 days of exposure that was 27-fold higher than that of the untreated controls. The total arginase activity also decreased 35, 52 and 82% after 4, 7 and 14 days of exposure and protein content decreased 57 and 65% after 7 and 14 days of exposure, respectively. Exposure to 6.67 p M arsenite also produced accumulation of intracytoplasmic lipid droplets, vacuolizations and enlargement of the intercellular spaces. On the other hand, exposure of hepatocytes to 1.33 pM arsenite caused an initial decrease of 20% in urea production, did not change cellular, released and total arginase activity and cellular protein content and produced accumulation of intracytoplasmic lipid droplets. These results show that long-term exposure of cultured rat hepatocytes to 6.67 pM arsenite decreases urea production, cellular and total arginase activity and protein content and increases the release of arginase into the culture medium. These alterations could be useful markers of hepatotoxicity in in vitro assays.