In the field of mycotoxin analysis, substantial problems shown by high between-laboratory standard deviations and noncomparable and nontraceable results have been caused by the lack of proper calibrants for external calibration. During a large-scale Standard Measurement and Testing project of the European Commission (EC) dealing with preparation and certification of reference materials for determination of the mycotoxin zearalenone (ZON) in maize, a ZON calibrant in acetonitrile was prepared and checked for purity, homogeneity, and stability. Before certification, on the basis of preparation, the calibrant was checked in a mini-interlaboratory study by UV spectrophotometric determination. The molar absorptivities of ZON in acetonitrile at 236, 274, and 314 nm were established, and as a main result, a common reference wavelength of 274 nm with molar absorptivity of 12623 +/- 111 L/mol x cm can be recommended for ZON in acetonitrile. A concentration and expanded uncertainty of the ZON calibrant of 9.95 +/- 0.08 microg/mL was calculated as a preliminary value before final evaluation through the certification panel of the EC.