The effects of different concentrations of indole-3-acetic acid (IAA) and benzyladenine (BA) on production of ajmalicine by multiple shoot cultures of Catharanthus roseus (C. roseus) were studied. By supplementing Murashige and Skoog's (MS) medium with a high concentration of IAA (11.42 muM) and a low concentration of BA (2.22 muM), shoot cultures accumulated high levels of ajmalicine. When culture medium was fortified with a low concentration of IAA (2.85 muM) and a high concentration of BA (8.90 muM), shoots released high levels of ajmalicine into the culture medium. Quantification of ajmalicine was performed by high performance liquid chromatography (HPLC). The highest concentration of ajmalicine production (0.166% dry wt) was obtained by shoot cultures grown in MS medium containing IAA (11.42 muM) on 20 days of cultivation. Shoot cultures accumulated ajmalicine 4.2-fold more in IAA (11.42 muM) supplemented medium compared with the high concentration of BA (8.90 muM). The content of ajmalicine concentration in the medium was quantified. Shoot cultures grown in BA (8.90 muM) supplemented medium released the maximum production of ajmalicine (0.853 g/L) into the culture medium after 15 days of cultivation. The experimental data show that the secretion of ajmalicine was 2-fold more into the culture medium supplemented with a high concentration of BA compared to that with a low concentration of BA. Data presented here show that production of ajmalicine by shoot cultures is not correlated with growth rate. Dimeric indole alkaloids vincristine and vinblastine were not present in shoot cultures. Ajmalicine production by shoot cultures was 2.4-fold higher compared to leaves of 1-year-old naturally grown plants.