Hirudin-1 is a highly selective inhibitor of thrombin secreted by the salivary glands of the medicinal leech Hirudo medicinalis. This direct anticoagulant is used for the treatment and prevention of disorders in the blood coagulation system. Apart from the existing recombinant analogue of hirudin-1 (63-desulfatohirudin-1) its modified analogues possessing higher activity and stability are of medical value. In this study artificial genes of hirudin-1 and two its analogues ([Leu1, Thr2]-hirudin-1 and [Leu1, Thr2]-hirudin-1/3) were synthesized and cloned in an expression vector pTWIN1 in frame with the gene of mini-intein DnaB from Synechocystis sp. Producing strains of the corresponding fusion proteins were constructed using E. coli strain ER2566. Biotechnological schemes for the production of 63-desulfatohirudin-1 and its analogues were developed. The scheme includes the following stages: isolation of the fusion protein, renaturation of the target protein incorporated into the fusion protein, pH-inducible cleavage of the fusion protein, and chromato-graphic purification of the target product. Antithrombotic activity of the peptides obtained was determined by a standard amidolytic assay. The developed methods for the production of 63-desulfatohirudin-1, [Leu1, Thr2]-63-desulfatohirudin-1 and [Leu1, Thr2]-63-desulfatohirudin-1/3 allowed us to obtain these peptides with high yields (14, 25 and 24 mg per 1 L of cell culture, respectively) and high activity (13423, 33333 and 19802 ATU/mg, respectively). © Pleiades Publishing, Ltd., 2012.