It has been known for a long time that mammalian peroxisomes are extremely fragile in vitro. Changes in the morphological appearance and leakage of proteins from purified particles demonstrate that peroxisomes are damaged during isolation. However, some properties of purified peroxisomes e.g. the latency of catalase, imply that their membranes are not disrupted. In the current study we tried to ascertain the mechanism of this unusual behavior of peroxisomes in vitro. Biochemical and morphological examination of isolated peroxisomes subjected to sonication or to freezing and thawing showed that the membrane of the particles seals after disruption restoring permeability properties. Transient damage of the membrane leads to the formation of peroxisomal 'ghosts' containing nucleoid but nearly devoid of matrix proteins. The rate of leakage of matrix proteins from broken particles depended inversely on their molecular size. The effect of polyethylene glycols on peroxisomal integrity indicated that these particles are osmotically sensitive. Peroxisomes suffered an osmotic lysis during isolation that was resistant to commonly used low molecular mass osmoprotectors e.g. sucrose. Damage to peroxisomes was partially prevented by applying more'bulky' osmoprotectors e.g. polyethylene glycol 1500. A method was developed for the isolation of highly purified and nearly intact peroxisomes from rat liver by using polyethylene glycol 1500 as an osmoprotector