Two cDNA clones encoding ribonuclease F1 (EC 3.1.27.3) have been isolated using a probe prepared by polymerase chain reaction with primers designed on the basis of amino acid sequence of the enzyme. They derived probably from the same gene and contained 393-base pair open reading frame encoding 131 amino acid residues (Mr 13,606) including a putative 25-residue signal peptide. The sequences of 43-base pair 5'-untranslated region and 125-base pair 3'-untranslated region including a poly(A) tail of 25 nucleotides were also elucidated. Homology analyses showed that cDNA for ribonuclease F1 has 65% homology to that for ribonuclease T1 in the coding region. At the preprotein level, they share 53% homology.