Publisher Summary DNA ligases refer to enzymes that catalyze the formation of a phosphodiester linkage between DNA chains. DNA ligases are essential reagents in studies on nucleic acid structure and metabolism. Their value derives from the specificity of the reaction and their ability to join polynucleotide chains covalently. In combination with polynucleotide kinase end-group labeling, DNA ligase can also be used to identify 3′-and 5′-end groups at single-strand interruptions by nearest neighbor analysis. DNA ligase was first identified in extracts of uninfected and T-phage- infected E. coli in 1967. This chapter focuses on the DNA ligases of E. coli and phage T4-induced enzymes. It considers studies on ligases from other sources, which supplement, or differ from those obtained with the E. coli and T4 enzymes. The chapter discusses the purification and physical properties of the ligases and outlines the properties and substrate specificities of the reactions catalyzed by the enzyme, including the intermediates in the reactions. It also highlights the in vivo roles of DNA ligases and describes the research applications of the enzyme..