The objective of this study was to investigate the relationship between the thiamine concentration and the fermentation patterns [pH, concentration of short-chain fatty acids (SCFA)] in the free liquid (FRL) and particle-associated liquid (PARL) of the rumen in dairy cows fed with graded concentrate levels in the diet. Four ruminally cannulated Holstein cows in mid lactation were fed [semi-ad libitum, 18 kg dry matter (DM)/day] diets consisting of hay (H) and slowly degradable concentrate (C), offered in five different H:C ratios (% DM basis) in the following sequence: period 1, 30:70; period 2, 40:60; period 3, 50:50; period 4, 60:40 and period 5, 75:25. A negative quadratic relationship was observed between thiamine concentration in FRL and intake of dietary thiamine (R(2) = 0.36), of concentrate (R(2) = 0.38) and of digestible non-fibre carbohydrates (R(2) = 0.37). The thiamine concentration in PARL was higher (p < or = 0.05) compared to FRL. The ruminal thiamine concentration correlated negatively to pH and positively to concentrations of SCFA and propionate in the rumen. R(2) of these relationships varied markedly (0.002 and 0.77), depending on time after feeding and fluid digesta compartment. The higher R(2) were observed at 8 and 11 h after the morning feeding for FRL (R(2) 0.46-0.71) and PARL (R(2) 0.41-0.77), respectively.