The need for new drugs to treat infections caused by antibiotic-resistant bacterial strains has prompted many studies to identify novel targets in pathogenic bacteria. Among the three DNA polymerases expressed by bacteria, one of these, designated pol III, is responsible for DNA replication and growth of bacteria and, therefore, warrants consideration as a drug target. However, the pol III enzymes of Gram-positive and Gram-negative species are quite different, and the Gram-positive enzyme pol IIIC has been more extensively studied as a drug target than the Gram-negative enzyme pol IIIE.DNA polymerases are unique enzymes with respect to the five substrates (four dNTPs, one of which is radiolabeled, and primer:template DNA) that they typically utilize. Variations of the assay, e.g., by leaving out one dNTP but allowing measurable incorporation of the remaining substrates, or use of homopolymer primer:templates, may be used to simplify the assay or to obtain mechanistic information about inhibitors. Use of gel analysis of primer extension assays can also be applied to study alternate substrates of DNA polymerases. Methods to isolate pol IIIC from Gram-positive bacterial cells and to clone and express the polC gene are described in this chapter. In addition, the assay conditions commonly used to identify and study the mechanism of inhibitors of pol IIIC are emphasized.