An international collaborative study was organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM) to establish the WHO second International Standard (IS) for amphotericin B as the stocks of the 1st IS, established in 1963, were close to depletion. The candidate material did not show any sign of potency loss when kept at elevated temperatures of + 4 degrees C, + 20 degrees C, + 37 degrees C and + 45 degrees C for 3 months. Ten laboratories from 8 countries participated in the collaborative study. Potencies of the candidate material were estimated by microbiological assays with sensitive micro-organisms. To ensure continuity between consecutive batches, the 1st IS for amphotericin B was used as the standard. Based on the results of the study, the 2nd IS for amphotericin B was adopted at the meeting of the WHO Expert Committee on Biological Standardisation (ECBS) in 2007 with an assigned potency of 944 International Units per mg (IU/mg). The 2nd IS for Amphotericin B is available from the EDQM.