Cultivation of the fungus Penicillium spp. HS-3, an endophytic fungus from the stems of Huperzia serrata, led to the isolation of a new diketopiperazine alkaloid, named as tryhistatin (1), together with three known ones, 2 - 4, all of which share a similar backbone. The structure of 1 was established by detailed interpretation of the 1D- and 2D-NMR spectra, and HR-ESI-MS data. Furthermore, 1D- and 2D-NMR experiments were employed to elucidate the structure of 2 which was deduced previously only on the basis of the LC/MS method. In this article, we confirmed the correctness of the previously reported structure and made a complete assignment of the NMR signals of 2 for the first time. © 2010 Verlag Helvetica Chimica Acta AG.