Tissue culture of Rauwolfia serpentina is an alternative source of more than 20 indole alkaloids that have antiarrhythmic, hypotensive, psychotropic, and anti-inflammatory effects. The paper was aimed at determining the hypotensive activity of five fractions (containing different combinations of alkaloids) of the extracts from the biomass of R. serpentina tissue culture (K-27M line). To achieve this aim the following methods were used: in vitro plant tissue culture, HPLC analysis of indole alkaloids, assessment of the effects of fractions on the smooth muscle of the isolated rat thoracic aorta in vitro and on the blood pressure after intravenous administration in vivo. The five fractions of the extract from cell biomass of R. serpentina (K-27M line) were isolated from dried or live biomass and differed in both qualitative and quantitative compositions. The direction of action and the magnitude of the effect were found to depend on the percentage composition of alkaloids in the fraction. It was shown that fraction 1 containing ajmaline and acetylajmaline (total alkaloids content of 2.2%) and fraction 2 containing ajmaline, raucaffricine, and acetylajmaline (total alkaloids content of 6.4%) exerted vasoconstrictor effects on thoracic aorta muscles. Fraction 4 containing vomilenine, acetylajmaline, methylajmalicine, ajmalicine, and rauffloridine (total alkaloids content of 65%) and fraction 5 containing mainly acetylajmaline with lesser amounts of vomilenine, methylajmalicine, and ajmalicine (total alkaloids content of 47.4%) were found to have a relaxing activity in vitro. Intravenous administration of fractions 4 and 5 resulted in a short-time (15–30 s) hypotensia, with blood pressure drop by 25% to 30%. All the 5 fractions of the extracts from Rauwolfia serpentina K-27M line demonstrated either vasoconstrictor or relaxing effects depending on the alkaloids type and content.