The cyanobacterial toxin cylindrospermopsin (CYN) is a frequent contaminant of freshwaters throughout the world, including those that are sources of drinking water. The first cases of human poisoning attributed to this toxin occurred from a treated drinking water supply in Queensland, Australia, in 1979. The toxin causes extensive damage to the liver, kidneys, spleen, heart, and other organs. It is known to be a potent protein synthesis inhibitor, but there is mounting evidence for genotoxicity and that it metabolizes to even more toxic forms. As part of a risk assessment process leading to a guideline for a safe drinking water level for this toxin, we performed a series of experiments to determine a no-observed-adverse-effect level (NOAEL) for this toxin. In the first trial male mice were exposed to CYN-containing cyanobacterial extract in their drinking water (0–657 μg CYN kg−1 day−1) for 10 weeks. In the second trial mice received purified CYN by daily gavage (0–240 μg CYN kg−1 day−1) for 11 weeks. Body and organ weights were recorded; urine, serum, and hematology analyses were performed; and histopathological examination of tissues was carried out. Body weights were significantly increased at low doses (30 and 60 μg kg−1 day−1) and decreased at high doses (432 and 657 μg kg−1 day−1). Liver and kidney weights were significantly increased at doses of 240 μg kg−1 day−1 and 60 μg kg−1 day−1, respectively. Serum bilirubin levels were significantly increased and bile acids significantly decreased at doses of 216 μg kg day−1 and greater. Urine total protein was significantly decreased at doses above 60 μg kg−1 day−1. The kidney appeared to be the more sensitive organ to this toxin. If it is assumed that increased organ weights and changes in functional capacity are responses to an underlying toxic effect, then the NOAEL based on this data is 30 μg kg−1 day−1, which, with standard calculations and uncertainty factors, provides a proposed guideline safety value of 1 μg/L in drinking water. © 2003 Wiley Periodicals, Inc. Environ Toxicol 18: 94–103, 2003.