Bovine seminal ribonuclease (BS-RNase), a homolog of bovine pancreatic ribonuclease (RNase A), is isolated as a dimer in which the subunits are cross-linked by two disulfide bonds. In addition to this anomalous quaternary structure, the enzyme has extraordinary biological properties, such as antispermatogenic, antitumor, and immunosuppressive activities. The molecular bases for these properties are well-suited for exploration with the techniques of recombinant DNA. Accordingly, a gene encoding BS-RNase was designed based on criteria expected to maximize the translational efficiency of its mRNA in Escherichia coli. This gene was constructed from 12 synthetic oligonucleotides and expressed with the phage T7 system. The protein thus produced was insoluble and accumulated under optimal conditions to 15% of total cellular protein or 200 mg/liter of culture. Ribonuclease activity was generated by air oxidation of the reduced and denatured protein. Three forms of active BS-RNase were isolated by gel filtration chromatography: the well-characterized dimer and monomer and a previously uncharacterized form that migrated as a trimer. The ribonuclease activities of all three forms were equivalent to or higher than that of dimeric BS-RNase isolated from bull seminal plasma.