Maturation, turnover, and quality control of RNA are performed by many different classes of ribonucleases. Escherichia coli RNase II is the prototype of the RNase II family of ribonucleases, a ubiquitous family of hydrolytic, processive 3'-> 5' exonucleases crucial in RNA metabolism. RNase R is a member of this family that is modulated in response to stress and has been implicated in virulence. In this work, RNase I I-like proteins were characterized in the human pathogens Salmonella typhimurium and Streptococcus pneumoniae. By sequence analysis, only one member of the RNase II family was identified in S. pneumoniae, while both RNase II and RNase R were found in Sa. typhimurium. These enzymes were cloned, expressed, purified, and characterized with regard to their biochemical features and modular architecture. The specificity of substrates and the final products generated by the enzymes were clearly demonstrated. Sa. typhimurium RNase II and RNase R behaved essentially as their respective E. coli counterparts. We have shown that the only hydrolytic RNase found in S. pneumoniae was able to degrade structured RNAs as is the case with E. coli RNase R. Our results further showed that there are differences with regard to the activity and ability to bind RNA from enzymes belonging to two distinct pneumococcal strains, and this may be related to a single amino acid substitution in the catalytic domain. Since ribonucleases have not been previously characterized in S. pneumoniae or Sa. typhimurium, this work provides an important first step in the understanding of post-transcriptional control in these pathogens.