A new endoribonuclease activity, RNase F, was partially purified from Escherichia coli cells. This activity can specifically cleave a precursor RNA molecule (of species 1) isolated from T4-infected cells [N. Watson & D. Apirion (1981) Biochem. Biophys. Res. Commun. 103, 543-551]. The cleavage results in products which are very similar to RNA molecules found in the cell, generating a 3'-phosphate and a 5'-hydroxyl groups. The cleavage takes place between a cytosine and an adenine moiety, within a possible loop and stem structure; the cut is in the border between the double-stranded and single-stranded regions of this structure. This specificity of this enzyme could be the introduction of a cleavage near the 3' ends of tRNA molecules and other RNAs like species 1 which could resemble tRNA in their three-dimensional structure.