Three Arg/Lys-Xaa bonds in the B-chain of human alpha-thrombin were found to be the major autolytic sites. Under the conditions of 1 mg of alpha-thrombin/ml in 50 mM-ammonium bicarbonate solution at 25 degrees C, the 50% cleavage times of Lys-Gly (residues 154-155), Arg-Tyr (residues 70-71) and Arg-Asn (residues 73-74) were 32 h, 72 h and 96 h respectively. Fragments generated from these three major autolytic sites were purified and analysed. In addition, minor and random autolytic cleavages occurred simultaneously that eventually led to the complete breakdown of the enzyme. These results reveal several novel aspects about the process of autolysis and the structure of autolysed human thrombin. It identifies a major autolytic site at Arg-Tyr (residues 70-71) that has not been previously reported. It demonstrates that beta-thrombin is not an obligatory intermediate during the process of conversion of alpha-thrombin into gamma-thrombin. There exists a new form of autolysed thrombin, designated as beta'-thrombin (with cleavage at Lys-Gly only), which also serves as the intermediate in the conversion of alpha-thrombin into gamma-thrombin. It shows that autolysis of human alpha-thrombin does not proceed in an absolutely clear-cut manner. Numerous minor cleavages, which amount to approx. 20% of the three major autolytic sites, occur simultaneously. It is the first time that several autolytic sites of human alpha-thrombin have been quantitatively analysed, and that it has been shown that formation of beta-, beta'- and gamma-thrombins can be quantitatively followed by the h.p.l.c. method. Furthermore, the data demonstrate that alpha-thrombin and the autolysed thrombin (mixture of beta-, beta'- and gamma-thrombins) have comparable proteolytic activity and specificity towards various sizes of non-fibrinogen polypeptide substrates with relative molecular masses ranging from 3000 to 25,000.