Central composite design was used to quantify the relationship between darkness, melatonin and serotonin with growth of Tetrahymena thermophila and hydrolytic enzyme activities. Circadian variation was the most important factor to optimise the maximal population without a long generation time; i.e. light at the beginning of culture for 640 min followed by darkness for 800 min. In addition, the medium should be supplemented with serotonin and melatonin (0.1 mM each). Supplemental melatonin and serotonin increased lipase, phosphatase and protease activities but without a circadian variation.