Sampling of intracellular metabolites in Mortierella alpina was investigated as part of a metabolomics study. After comparison of four sampling protocols, rapid filtration of the culture using a laboratory-made nylon filter and absorbent gauze under normal pressure followed by quenching in liquid N-2 and grinding (the improved protocol) was the most effective. Rapid filtration under normal pressure decreased intracellular metabolites leakage and subsequent grinding of cells contributed to intracellular metabolites extraction. The above quenching method together with 75 % (v/v) ethanol, buffered with 60 mM HEPES, at 80 degrees C for 3 min is therefore suitable for sampling intracellular metabolites in M. alpina.