Antibody against ribonuclease (RNase) F1 was raised in a rabbit by intramuscular injection of the protein with Freund’s complete adjuvant. Excess amount of the antibody totally inhibited the enzyme activity on RNA substrate. Quantitative precipitin analysis showed the presence of at least 4 or 5 epitopes (antigenic determinants) on the RNase F1 molecule. Cross-reactivity of the antibody with RNases T1, Ms, U2, St and T2 was examined. Although the double diffusion test detected no cross-reactivity with any of these RNases, Western blot analysis with anti RNase F1 antibody revealed weak cross-reactivity with RNases Ms and T1.