Lysosomal acid lipase (LAL) gene mutations were identified in three patients with cholesteryl ester storage disease (CESD). Direct sequencing of genomic DNA revealed that: patient 1 was a compound heterozygote for a P181L mutation and an A to G3' splice site substitution that causes skipping of exon 7, with a loss of 49 amino acids from LAL (delta 205-253); patient 2 was a compound heterozygote for a G66V mutation and a 5' splice site mutation (G to A) that leads to skipping of exon 8 (delta 254-277); and patient 3 was a compound heterozygote for a L273S mutation and an unidentified null allele. Furthermore, patients 2 and 3 showed a novel G-2A polymorphism that could be detected by an Xbal restriction fragment length polymorphism. All these mutants and a previously reported H274Y allele were expressed in vitro in HeLa cells using the vaccinia T7 expression system. The resulting recombinant proteins were inactive towards cholesteryl oleate and trioleylglycerol, demonstrating the direct involvement of these mutations in the pathogenesis of CESD. Immunoblotting of normal LAL expressed in HeLa cells revealed four major molecular forms, at least two of high molecular mass (54 and 50-51 kDa) and two of low molecular mass (42 and 43 kDa). L273S and P181L substitutions and delta 254-277 were shown to result in altered LAL molecular forms, some of which suggest that post-translational processing may interfere with the catalytic activity of LAL.