The objective of this study was to investigate the pretreatment effects of high-fibre substrate on particle size distribution in a full-scale agricultural biogas plant (BGP). Two digesters, one fed with pretreated material and one with untreated material, were investigated for a period of 90 days. Samples from different positions and heights were taken with a special probe sampling system and put through a wet sieve. The results show that on average 58.0 +/- 8.6% of the particles in both digesters are fine fraction (<0.063 mm). A higher amount of particles (13.1%) with a length >4 mm was measured in the untreated digester. However, the volume distribution over all positions and heights did not show a clear and uniform distribution of particles. These results reveal that substrate pretreatment has an effect on particle size in the fermenting substrate, but due to the uneven distribution mixing, is not homogeneous within the digester. (C) 2014 Elsevier Ltd. All rights reserved.