Rotifer contamination, as a severe constraint in mass cultures, impedes industrial-scale cultivation of microalgae. Once contamination occurred, the rotifers released something that inhibited microalgal cell growth in addition to algal reduction due to predation. With the aim of further identifying the type and effects of the inhibitor released from rotifers, Chlorella sp. was cultured in fresh modified F/2 medium and in media containing various proportions of boiled rotifer culture-medium filtrate (RCF), as well as in media containing water-soluble or lipid-soluble fractions isolated from RCF. Then, the growth and photosynthesis of Chlorella sp. were measured. The rotifer inhibition of Chlorella growth could not be eliminated by boiling the RCF, indicating that the inhibitor was not likely a protein. The growth and photosynthesis of Chlorella sp. was not inhibited by the water-soluble RCF fraction, but they were significantly inhibited by the lipid-soluble RCF fraction, in a dose-dependent manner. Further, the lipid-soluble fraction decreased energy conservation and photosynthetic electron transport, which induced a severe decrease in PSII activity and a decrease in the net photosynthetic O 2 evolution rate. Based on these physiological responses of Chlorella cells, the lipid-soluble fraction rather than the protein or water-soluble fractions was determined to contain the responsible inhibitor, suggesting the direction of further studies .