AFPA culture medium, which is used for recognition of Aspergillus flavus and A. parasiticus, has been validated in a collaborative study including nine laboratories located in Australia, Brazil, Denmark, The Netherlands, Sweden and United Kingdom. Three freeze-dried fungal mixtures, containing A. flavus/A. parasiticus and background fungi, were produced and checked for homogeneity. The coefficients of variance were low, ranging from 0.81% to 1.09% for total fungal counts and between 2.50% and 2.72% for counts of A. flavus/A. parasiticus. The laboratories analysed the contents of two vials of each mixture on commercial A. flavus and A. parasiticus agar (AFPA), in-house-made AFPA, and on a standard media, dichloran 18% glycerol agar (DG18). Reproducibility values for counts of A. flavus/A. parasiticus indicated no differences between the commercial AFPA and the in-house-made AFPA. Variation between laboratories was low, indicating that the medium was effective in use. Reproducibility values for DG18 were higher. There were no differences in counts of A. flavus/A. parasiticus on AFPA and DG18. However, DG18 gave slightly higher total fungal counts compared to AFPA.