Biomass and secondary metabolite accumulation were assayed on a wide range of auxins, cytokinins and their combinations using Ecballium elaterium callus tissue. The best combination was that consisting of alpha-naphthalene acetic acid (NAA) and benzylamino purine (BAP) (1 mg/l of medium, each). NAA and BAP were set up in a grid to determine concentrations that produced the best callus as regards fresh and dry weights and yield of cucurbitacins, particularly cucurbitacin E. The best combination for callus proliferation consisted of 5 mg/l NAA and 5 mg/l BAP, reaching a peak at week 5. For cucurbitacin production, an optimum yield was obtained at week 3 with 5 mg/l NAA.