Multiple quantitative trait locus (QTL) mapping studies designed to localize seizure susceptibility genes in C57BL/6 (B6, seizure resistant) and DBA/2 (D2, seizure susceptible) mice have detected a significant effect originating from midchromosome 5. To confirm the presence and refine the position of the chromosome 5 QTL for maximal electroshock seizure threshold (MEST), reciprocal congenic strains between B6 and D2 mice were created by a DNA marker-assisted backcross breeding strategy and studied with respect to changes in MEST. A genomic interval delimited by marker D5Mit75 (proximal to the acromere) and D5Mit403 (distal to the acromere) was introgressed for 10 generations. A set of chromosome 5 congenic strains produced by an independent laboratory was also studied. Comparison of MEST between congenic and control (parental genetic background) mice indicates that genes influencing this trait were captured in all strains. Thus, mice from strains having D2 alleles from chromosome 5 on a B6 genetic background exhibit significantly lower MEST compared with control littermates, whereas congenic mice harboring B6 chromosome 5 alleles on a D2 genetic background exhibit significantly higher MEST compared with control littermates. Combining data from all congenic strains, we conclude that the gene(s) underlying the chromosome 5 QTL for MEST resides in the interval between D5Mit108 (26 cM) and D5Mit278 (61 cM). Generation of interval-specific congenic strains from the primary congenic strains described here may be used to achieve high-resolution mapping of the chromosome 5 gene(s) that contributes to the large difference in seizure susceptibility between B6 and D2 mice.