Totally reduced and denatured seminal ribonuclease was regenerated using the glutathione redox system. The refolding kinetics of the enzyme were determined as a function of redox state, temperature from 14 to 43 degrees C, pH, and protein concentration. The maximal rate of regeneration occurred with 3 x 10(-3) M reduced glutathione, 6 x 10(-4) M oxidized glutathione, 24 to 30 degrees C, and pH 8.2. The products of the refolding process were characterized by Sephadex G-75, sodium dodecyl sulfate gel electrophoresis, enzymatic activity, circular dichroism, and amino acid analysis. The results indicate that the native dimeric form of the enzyme is not produced during refolding to any appreciable extent; rather, the major product is monomeric. The purified monomer exhibits twice the activity of the native enzyme toward yeast RNA. Its circular dichroism spectrum is different from the native enzyme and is quite similar to that of pancreatic ribonuclease A. Amino acid analyses showed that two glutathione molecules are bound to the monomer, suggesting that cysteine-31 and -32, which normally form the intermolecular disulfide bonds, are blocked.