Niemann–Pick disease type C (NP‐C) is a rare genetic disorder characterized by progressive neurodegeneration, frequent developmental delay and early death. Tissues of affected individuals accumulate large quantities of free cholesterol in lysosomes. Because cytotoxic oxygenated derivatives of cholesterol are known to form readily when cholesterol concentrations are elevated, we searched for these compounds in liver, kidney, spleen and brain from mice with the NP‐C phenotype. In order of abundance, we identified 7α‐ and 7β‐hydroxycholesterol, 5α,6α‐epoxycholestan‐3β‐ol, 4β‐hydroxycholesterol, cholest‐4‐en‐3β,7α‐diol and cholest‐4‐en‐3β,6β‐diol in most tissue samples. Cholesterol concentrations in affected mice were increased 3‐fold in kidney and 7‐ to 8‐fold in spleen and liver compared to controls (all p<0.001) but were unchanged in brain. Although oxysterol levels were markedly elevated in non‐brain tissue, the oxysterol and cholesterol concentrations increased proportionally so that oxysterols expressed as percentage of total sterols were the same for all animals (0.34±0.19% averaged over all organs in affected animals vs 0.40±0.42% in control mice). In contrast to peripheral tissue, we could not detect any increase in either absolute or relative oxysterol levels in the brains of affected and control mice (49±61 vs 53±43 μg/g wet weight and 0.45±0.52 vs 0.47±0.37%, respectively). Thus, brain sterols are normal in NP‐C mice and it is unlikely that an accumulation of cytotoxic oxygenated derivatives of cholesterol could account for the progressive neuropathology seen in the disease.