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This article presents the data of studies on optimization of conditions for culturing the causative agent of bovine adenovirus infection on continuous cell culture lines. The work used the reference strain of bovine adenovirus subgroup 1 “Adeno III WBR - 1”; cell lines of newborn Syrian hamster kidney (BHK-21/13), calf kidney (Taurus-1), endothelium of coronary vessels of the embryo cow (KST), epithelium of the lung embryo of cattle (LEK) and Madin-Darby bovine kidney (MDBK). It was found that with a stationary cultivation method, the adenovirus strain quickly adapted to the continuous cell culture lines BHK-21/13, Taurus-1, KST and MDBK. Among the continuous cell cultures, the MDBK (0.0001 MOI/cell) demonstrated the highest sensitivity to infection with the adenovirus strain. The maximum accumulation of the adenovirus strain in the culture fluid was observed 24 hours after infection. Comparative assessment of the level of strain accumulation in the culture fluid obtained using the MDBK cell line after various methods of its cultivation showed that the maximum titer of the virus strain ((6.55±0.21) lg TCID50/cm2) was observed with the roller method of cell cultivation. The obtained results can be used to develop a test system for the purpose of identifying pathogens of adenovirus infection in cattle.