A mutant of Salmonella typhimurium has been isolated that has less than 5% of the ribonuclease activity of the parent strain. Mutant screening and enzyme assays were done in the presence of ethylenediaminetetraacetic acid, a substance that activates ribonuclease I and inhibits other known microbial nucleases. Genetic mapping indicates that the mutation is located between the purE and gal genes on the Salmonella chromosome. A ribonuclease-deficient mutant that carries a deletion in the pyrF gene is unable to utilize ribonucleic acid as a pyrimidine source, whereas the pyrF parent with normal ribonuclease activity will grow. This suggests that the enzyme may perform a scavenge function in the utilization of exogenous ribonucleic acid. Loss of this enzyme seems to have no detrimental effects on the growth of Salmonella.