Three ways to calculate average hyphal cross section in agarfilm slides were compared in a field study including 5 soil horizons sampled 4 times. The method of calculation strongly affected the estimated value for soil fungal biovolume. Biovolume estimated with the different methods varied by more than 30%. Great differences in average cross section were found between the soil horizons, stressing the importance of using proper field measurements for biovolume estimations. The average cross section did not vary significantly between the sampling occasions in identical soil layers.