Four of five mutations producing GPI1 null lethal phenotypes in the homozygous state, which were previously identified from the offspring of male mice, spermatogonially treated with N-ethyl N-nitrosourea (ENU), have been characterized at the nucleotide level by reverse transcription of RNA from heterozygotes for mutant and wild-type alleles and cycle sequencing with cDNA-derived primers. In three of the mutations studied, a single nucleotide substitution, altering the predicted amino acid on translation, was observed in the mutant allele. In Gpi1-sam1H amino acid residue 277, TCA Ser (wild type), is altered to CCA Pro, and in Gpi1-sbm3H and Gpi1-sbm4H amino acid residue 510 Asp GAC (wild type) is altered to GGC Gly. These ENU-induced mutations occur at A-T base pairs in agreement with the current view of the mechanism of action for this mutagen. These changes also occur at residues implicated as being important in the catalytic functioning of the enzyme, from crystallographic studies, and may explain the loss of enzyme function. The fourth identified mutation, Gpi1-sbm2H, is a deletion of amino acid residues Arg134 to Leu162 inclusive, which may arise from incorrect splicing of mRNA; a fifth mutation has remained undetermined.