Four ENU-induced mutations were previously identified at the triosephosphate isomerase (TPI) locus in mouse germinal mutation experiments. Each of the mutants is associated with a 50% loss of enzymatic activity in the F1 (heterozygous) animals. Exons of the TPI gene from control mice and heterozygous mutant mice were PCR amplified and sequenced as necessary to determine the molecular lesion in the mutant alleles. Mutants Tpi*M-1NEU and Tpi*M-2NEU carried the same T:A to A:T transversion in exon 6, resulting in a Leu to Gln substitution at residue 192. Amino acid residue 192 is located in alpha-helix H6 of the protein. Tpi*M-4NEU contained a T:A to A:T transversion within the codon for residue 162 in exon 5, also causing a Leu to Gln substitution. This mutation is located at the beginning of beta-strand B6, within a highly conserved sequence region surrounding the active site residue Glu 165. Sequence analysis of Tpi*M-3NEU revealed an A:T to C:G transversion, changing the stop codon to a codon for Cys, with the resulting addition of 19 predominantly hydrophobic amino acids to the protein. All four mutations occurred at an A:T base pair. In each case, the mutation site was flanked on both sides by G:C base pairs. Each of the sequence alterations has a potential impact on the structure of the TPI protein that is consistent with the existence of a null allele. In addition to providing insight into the molecular basis of ENU induced germ cell mutations and the differences in mutation spectra among organisms, these mutants represent models for structure-function studies of this highly conserved enzyme.