We have determined that a functional gene coding for ribonuclease H seems to be essential for cell growth in Escherichia coli. A strain was made with two copies of the rnh gene by lysogenizing an E. coli strain with a lambda phage bearing a copy of the rnh gene. Inactivation of one of the two copies of the rnh gene was accomplished by transformation with a linear DNA molecule that had the gene for chloramphenicol acetyltransferase inserted near the middle of the rnh gene. In recombinants that had an inactive gene replacing the normal chromosomal rnh gene, the lambda rnh prophage supplies an intact functional copy of the rnh gene. Curing the cells of the lambda rnh prophage left the cell with an inactive rnh gene and resulted in cell death. An intact functional rnh gene provided on a plasmid permits normal curing, and cured survivors were readily obtained. The technique described is probably generally applicable for assessing the requirement for other E. coli genes.