Pharmacokinetics of the promising antitumor peptide HLDF-6-AA (Ac-ThrGlyGluAsnHisArg-NH2) was studied using its uniformly tritiated derivative. Experiments were performed on male Wistar rats, Balb/c mice and Chinchilla rabbits. The tritium labeled peptide [3H]HLDF-6-AA with the molar radioactivity of 50 Ci/mmol was obtained by the reaction of high-temperature solid-state catalytic isotope exchange (HSCIE). Under intravenous bolus administration of HLDF-6-AA peptide to rats and rabbits, the characteristics of its pharmacokinetic profile in the blood were obtained and the values of the main pharmacokinetic parameters of HLDF-6-AA peptide, and its active metabolite HisArg-NH2 were calculated. It was shown that parameters of the retention time in the body and the rate of elimination for peptides HLDF-6-AA and HisArg-NH2 in rats and rabbits are close and in rats they are about 7 and 21 min, respectively. It was shown that repeated administration of the drug does not lead to a change in its regular cumulation and does not cause a change in its pharmacokinetics compared with a single administration. The linearity of the dependence of pharmacokinetic parameters on the amount of administered peptide in the range of 2–22 mg/kg was proved in experiments in rats. As a result of the study of the distribution of HLDF-6-AA peptide and its metabolite HisArg-NH2 between the blood and peripheral tissues of mice, it was shown that the maximum concentration of HLDF-6-AA peptide is observed in the kidney tissues and a somewhat smaller concentration in the omentum. It was found that 15–20 min after intraperitoneal administration of HLDF-6-AA to mice, the concentration of HisArg-NH2 peptide begins to exceed the concentration of HLDF-6-AA peptide, which is caused by its greater resistance to proteolytic hydrolysis. The highest concentration of HisArg-NH2 peptide is observed in the kidney and liver tissues.