A yellow optically-active pigment, chrysophysarin A (1), has been isolated from microplasmodia of the slime mould Physarum polycephalum. The structure was established by means of 1D and 2D NMR spectroscopy and mass spectrometry. The absolute configuration was assigned by the synthesis of N-(3,3-dimethylacryloyl) derivatives of (S)- and (R)-leucine and by comparison of the corresponding CD spectra. The biosynthesis of the compound was elucidated by feeding labeled acetate to the plasmodia.