Capsaicin synthesis by suspension cultured cells of Jalapeno pepper (Capcicum annuum L.) was assessed in vitro under various conditions including temperature (23 and 30℃), light intensity (with light and without light), and shaking speed (110 and 200 rpm). Capsaicin production increased, while the cell biomass growth decreased possibly due to the production of a secondary metabolite. Capsaicin synthesis was primarily affected by light condition. Cells cultivated at 110 rpm and 23℃ under light condition yielded the highest fresh weight, while those cultivated under the same condition, but without light resulted in the lowest cell mass. Capsaicin content in cells of 18-day-old pepper grown at 110 rpm and 23℃ under light was 0.125% of the cell mass. However, without light treatment, the capsaicin content in cells at the same shaking speed and temperature increased up to 169%, indicating no light is favored in the capsaicin synthesis by Jalapeno pepper. Increasing the shaking speed from 110 to 200 rpm without light enhanced the capsaicin synthesis. Results of this study demonstrate that light condition is the limiting factor in the synthesis of capsaicin in tissue-cultured Jalapeno pepper cells.