A ribonuclease has been purified to homogeneity from bovine seminal plasma by precipitation with 50-70% saturated ammonium sulfate, followed by chromatographies on concanavalin A-Sepharose 4B, DEAE-cellulose, agarose-5`-(4-aminophenylphospho)-uridine 2`(3`)-phosphate, and Sephadex G-75. The homogeneity of this ribonuclease was confirmed by polyacrylamide gel electrophoresis. Molecular weight for this purified ribonuclease was 12,500 as estimated by gel filtration. The enzyme activity was activated by Na^+, K^+, Mg^(2+), Ba^(2+), Fe^(2+), and EDTA and inhibited by Ca^(2+), Mn^(2+), Zn^(2+), and Cu^(2+). The purified ribonuclease preferentially hydrolyzed poly(U) over poly(C) and yeast RNA. The N-terminal amino acid sequence of 31 residues for the purified seminal ribonuclease has been determined by automatic sequencing of the native protein. The following sequence was deduced: Val^1 Asp Ser Lys Gly^5 Gly Lys Tyr Gln Arg^(10) Glu His Met Asp Leu^(15) Asp Ser Leu Pro Ala^(20) Ala Bil Val Gly Thr Tyr^(25) Cys Asp Ala Met Lys Leu These results suggested that the purified bovine seminal ribonuclease was a prostate-specific enzyme.