A general procedure has been developed for the simple and rapid construction of circularly permuted proteins. The polymerase chain reaction (PCR) was utilized to amplify the gene for ribonuclease T1 (RNase T1) in two separate pieces, and these fragments were recombined in reverse order to create the circularly permuted gene. The circularly permuted analog of RNase T1 was assembled by first removing the disulfide bond between Cys-2 and Cys-10 through substitution with alanine residues to create the mutant (C2A, C10A). The original amino- and carboxyl-terminal ends of (C2A, C10A) were then covalently linked with the peptide Gly-Pro-Gly, and new termini were introduced between residues Gly-34 and Ser-35. The bacterially expressed and circularly permuted variant of RNase T1, cp35S1, was 28% as active as the native enzyme in the catalytic hydrolysis of RNA, and thus the mutant protein must fold to a conformational state quite similar to that of the native enzyme. Amino acid sequence analysis and mass spectrometry have confirmed the primary structure of the reconstructed protein. Thermodynamic stabilities at pH 5.0 of wild-type RNase T1, (C2A, C10A), and cp35S1 proteins were found to be 10.1, 6.4, and 4.2 kcal/mol, respectively.