3-Nitropropionic acid (NPA) is a known toxic metabolite produced by many species of Astragalus of the Leguminosae family. In 1992, Liu et al. reported that some species of Arthrinium were the etiological fungi of moldy sugarcane poisoning (MSP) occurring in China. The toxic metabolite NPA produced by Arthrinium was the main causative agent. Ten percent (88/884) of the MSP patients died, and many were left with lifelong disabilities. In this study, an accurate, sensitive, and rapid method for quantifying NPA produced by Arthrinium species in sugarcane juice was developed. The method includes extraction, thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC). Fourteen strains of Arthrinium obtained from the American Type Culture Collection (ATCC) were examined for NPA-producing ability. Eight strains (57.1%), including three of A. sacchari, two of A. phaeospermum, and one each of A. terminalis. A. aureum, and A. sereaensis, were positive for NPA production. The highest level of NPA produced by A. sacchari ATCC 76981 was 1.7 mg/ml when cultured at 24-degrees-C for 2 weeks. The optimum temperature for the production of NPA toxin was 24-degrees-C. The optimum pH was around pH 5.0. NPA production was higher in stationary cultures than in those shaken at 120 rpm.