We report a defect in splicing of precursor messenger RNA (pre-mRNA) resulting from a naturally occurring mutation of the gene encoding purine nucleoside phosphorylase (PNP) in a patient with PNP-deficient severe combined immunodeficiency. This defects results from a G to T transversion at the terminal nucleotide of exon 2 within the 5' splice site of intron 2 and causes skipping of exon 2 during processing of PNP pre-mRNA. Translation of the misspliced mRNA results in a reading frameshift at the exon 1-exon 3 junction. The predicted polypeptide encoded by the aberrant mRNA is severely truncated, terminating at 31 amino acids. Only 4 residues at the NH2 terminus of the polypeptide correspond to PNP amino acids. Otherwise the translation product of the misspliced mRNA differs completely from PNP in amino acid sequence and has no PNP activity. The finding of exon skipping in PNP is the first report of a splicing defect resulting in PNP-deficient severe combined immunodeficiency. Analysis of the genomic context of the G-1 to T mutation of the 5' splice site lends support for the exon definition model of pre-mRNA splicing and contributes to the understanding of splice site selection.