The catfish, Plotosus lineatus, forms a dense ball-shaped school soon after hatching. The involvement of a chemical cue(s) in this behavior was indicated from the observations that P. lineatus was attracted not only to seawater conditioned with the schoolmates (schoolmate seawater) but also to the skin mucus collected from the schoolmates. To determine the nature of the chemical cue, we first established a novel bioassay that monitored a characteristic ‘turn behavior' toward an agar block containing skin mucus collected from the schoolmates. The bioassay-guided fractionations of skin mucus led to a final preparation wherein activity was contained in a single peak in high performance liquid chromatography on a polyamine column. The spectral data of the final preparation indicated that the purified material was a mixture of phosphatidylcholine molecular species, which was supported by the fact that the final active preparation lost the activity when treated with phospholipase A₂, indicating that the school recognition substance is degraded by phospholipase A₂. From these results, we proposed that the chemical cue to recognize the school in P. lineatus may be PC molecular species.