Nine domestic wine samples collected from a Japanese winery were examined for the presence of fumonisin B-1(FB1), fumonisin B-2(FB2), and fumonisin B-3(FB3), as well as ochratoxin A (OTA) and ochratoxin B (OTB). Wine samples spiked with(13)C-labeled internal standards (C-13(34)-FB(1)and(13)C(20)-OTA) were diluted with phosphate buffered saline (PBS) buffer, loaded on immunoaffinity cartridges to purify of fumonisins and ochratoxins, and subjected to liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis. The data revealed that the domestic wine samples were possibly contaminated with FB(1)and FB3, in addition to FB2, whereas none of the tested wine samples were contaminated with OTA and OTB. These results suggest thatFusariumfungi can be associated with the fumonisin contamination of Japanese domestic wine, whereasAspergillus nigerseems to be frequently reported as the major causal fungus of fumonisin contamination of wine in Europe. Analysis of the intermediate samples during the wine brewing indicated that fumonisin concentrations did not increase during wine production, suggesting that fumonisin contamination did not occur during the brewing process, but was derived from the raw materials of grape berries.