Escherichia coli ribonuclease LS is a potential antagonist of bacteriophage T4. When the T4 dmd gene is defective, RNase LS cleaves T4 mRNAs and antagonizes T4 reproduction. Our previous work demonstrated that E. coli rnlA is essential for RNase LS activity. Here we show that His-tagged RnlA cleaves T4 soc RNA at one of the sites also cleaved by RNase LS in a cell extract. The cleavage activities of His-tagged RnlA and the RNase LS activity in a cell extract were inhibited by Dmd encoded by T4 phage. Fractionation of the RNase LS activity in a cell extract showed that it sedimented through a sucrose density gradient as a 1000-kDa complex that included RnlA. Pull-down experiments revealed more than 10 proteins associated with His-tagged RnlA. Among these, triose phosphate isomerase exhibited a remarkable affinity to RnlA. These results suggest that RnlA plays a central role in RNase LS activity and that its activity is regulated by multiple components.