The thiamine concentration was studied in solid and fluid rumen digesta of three Holstein cows, fitted with rumen cannulae and fed grass silage-based TMR with three particle sizes (25, 11 and 5.5 mm). The decrease of the particle size led to an increase of the thiamine concentration in solid (P<0.05) and fluid rumen digesta. Thiamine concentration was increased (R²=0.56, P<0.001) as digestible organic matter intake increases. The pH (mean 5.81) did correlate negatively (R²=0.21 to 0.26, P<0.001) to the thiamine concentration of rumen. Thiamine concentration of the rumen digesta did reflect in great extent the changes of rumen conditions.