Genetic analyses of an Escherichia coli K-12 mutant possessing the amber mutation lig-321 were carried out. This mutant is defective in deoxyribonucleic acid (DNA) ligase and conditionally lethal. We constructed strains harboring an F'lig+ or F'lig-321 plasmid. Genetic complementation analyses were done by using these plasmids and by constructing a lig-4/F'lig-321 merodiploid. It was shown that lig-321 does not complement lig-4, unless the former is suppressed by an amber suppressor. The same was found to be the case between lig-321 and lig-ts7. Transductional mapping of lig-321, by a four-factor cross, revealed that lig-321 is very closely linked to lig-4. The frequency of recombinants between the two alleles was not unreasonable for assuming that they arose by intragenic recombination. The lig-4 and lig-ts7 alleles are known to reside in the structural gene for DNA ligase, in which lig-321 may also be located.