Niemann-Pick disease, an autosomal recessive lysosomal storage disorder, is caused by deficiency of acid sphingomyelinase. Sequence analysis of mRNA and genomic DNA of fibroblasts of a type A patient showed a single G1729 to A nucleotide transition. This mutation resulted in a substitution of serine for normal glycine at position 577 of the peptide sequence. Amplification of the genomic DNA region around the mutation and subsequent sequencing yielded exclusively the same base change found at the cDNA level. Expression studies with this abnormal cDNA in COS-1 cells revealed a complete loss of enzymatic activity of the mutated protein. These findings indicate that this mutation is responsible for the clinical disease of the patient.