Lysosomal acid lipase (LAL) is essential for the hydrolysis of the triglycerides and cholesteryl esters in lysosomes. Its deficiency produces two phenotypes, a severe infantile-onset variant, Wolman disease (WD), and a later onset variant, cholesteryl ester storage disease (CESD). A mouse model with a LAL null mutation was produced by targeting disruption of the mouse gene. Homozygote knockout mice (lal⁻llal⁻) produce no LAL mRNA, protein or enzyme activity. The lal⁻llal⁻ mice are born in Mendelian ratios, are normal appearing at birth, and follow normal development into adulthood. However, massive accumulation of triglycerides and cholesteryl esters occurs in several organs. By 21 days, the liver develops a yellow-orange color and is ∼1.5–2.0× larger than normal. The accumulated cholesteryl esters and triglycerides are ∼30-fold greater than normal. The lal⁺llal⁻ mice have ∼50% of normal LAL activity and do not show lipid accumulation. Male and female lal⁻llal⁻ mice are fertile and can be bred to produce progeny. This mouse model is a phenotypic model of human CESD, and a biochemical and histopathologic mimic of human WD. The lal⁺llal⁻ mice provide a model to determine the role of LAL in lipid metabolism and the pathogenesis of its deficiency states.