Protease Do in Escherichia coli is a cytoplasmic serine protease that has an unusually high molecular weight. This enzyme has also been found to be responsible for the hydrolysis of the ada gene product. Using the lambda gtll genomic library of E. coli, we isolated a 2.2 kb clone that overproduces protease Do. Expression of the protease Do gene (ptd) was not affected by addition of IPTG, and therefore the 2.2 kb clone should contain its own promotor sequence. Upon transformation of 2.2 kb DNA that had been disrupted by kan gene, ptd gene in chromosome was disrupted. The resulting mutant cell lacked protease Do and grew much slowly than its wild type. Thus, protease Do appears to affect normal cell growth perhaps by its proteolytic function but not essential for cell`s survival. Determination of the primary structure of the ptd gene and physiological role of protease Do are under investigation (Supported by a grant from the Ministry of Education).