A strain derived from a colony of BALB/c mice at the National Center for Toxicological Research, Jefferson, AR, USA (NCTR-BALB/c) suffers from an autosomal recessive disorder characterized by proliferation of secondary lysosomes with accumulation ofunesterified cholesterol in several tissues. The unesterified cholesterol content of spleens and lungs from the affected mice were elevated 8- and 3-fold respectively over age- and sex-matched controls. Postnuclear supernatants of tissue homogenates were fractionated by sucrose density gradient centrifugation and the fractions were analyzed for unesterified cholesterol, protein and marker enzyme activities for lysosomes (N-acetyl-beta-D-glucosaminidase, beta-D-glucuronidase), plasma membrane (alkaline phosphodiesterase I), endoplasmic reticulum (glucose-6-phosphatase) and mitochondria (cytochrome oxidase). The enzyme distribution profile showed that lysosomes of affected tissues floated at low density regions (density 1.05-1.08) of the gradient and contained substantial amount of tissue unesterified cholesterol. These low density lysosomes were purified about 17-fold (58% yield) from spleen and about 6-fold (32% yield) from lungs with minimal contamination by other organelles They were mostly intact as judged by high latency for N-acetyl-beta-D-glucosaminidase activity (70-100%). Lysosomes of control tissues were not found at the low density regions. The distribution profiles for other organelles were similar between affected and control tissues. Phospholipid composition of low density lysosomes were distinctly different from their respective tissue homogenates. Spleen and lung lysosomes were enriched in sphingomyelin and phosphatidylcholine respectively. The results suggest that these lysosomes acquire their low densities due to accumulation of unesterified cholesterol, the retention of which may be aided by sphingomyelin and phosphatidylcholine content of the lysosomes.