Cholesteryl ester storage disease (CESD) and Wolman disease are both autosomal recessive disorders associated with reduced activity and genetic defects of lysosomal acid lipase (LAL). We provide evidence that the strikingly more severe course of Wolman disease is caused by genetic defects of LAL that leave no residual enzyme activity. In a CESD patient, a G --> A mutation at position -1 of the exon 8 splice donor site results in skipping of exon 8 in 97% of the LAL hnRNA originating from this allele, while 3% are spliced correctly, resulting in full-length LAL enzyme. The mutant LAL mRNA codes for a protein lacking amino acids 254 to 277. On the other allele, a G --> T mutation leads to a premature stop codon at Gly245, resulting in inactive LAL enzyme. In addition, the previously identified Leu179 --> Pro mutation is present on this allele, and the LAL mRNA is rendered unstable by the premature stop codon. Analysis of two children with Wolman disease showed that both were homozygous for a G --> A mutation at position +1 of the same splice donor site as for the CESD patient, leading to skipping of exon 8. In contrast to the CESD patient, no correctly spliced mRNA was detectable. We have also expressed a wildtype LAL cDNA and the mutant LAL cDNA from one Wolman patient in Sf9 and H5 insect cells. We demonstrate that the LAL enzyme generated from the wildtype LAL cDNA was active in homogenates from Sf9 and H5 cells, while the enzyme with the internal deletion of 24 amino acids originating from the LAL cDNA of the Wolman patient was not. The combined data provide evidence that the only functionally relevant genetic difference between the Wolman patients and the CESD patient is that the splice defect in Wolman, which affects one of the invariable nucleotides of the splice consensus sequences (position +1), does not permit any correct splicing, whereas the defect observed in CESD (position -1) allows some correct splicing (3% of total LAL mRNA) and therefore the synthesis of functional enzyme.