An investigation was made of the effects of variation of the hydraulic retention time (HRT) and the concentrations of three different carbon sources (succinic acid, ethanol and acetic acid) on the denitrification activity of immobilized Pseudomonas butanovora cells. The highest denitrification activity was in all cases measured at a C: N ratio of 6: and a relatively low HRT (2.5 h). The highest denitrification rates were 1.17 kg NO3--N m(-3) d(-1) for succinic acid, 1.63 kg NO3--Nm(-3) d(-1) for ethanol and 1.53kg NO3--Nm(-3) d(-1) for acetic acid. At the same C:N ratios, ethanol and acetic acid proved to be better substrates for the reduction of nitrate and nitrite. The determined optimum C: N ratios were 1.78 +/- 0.31, 0.95 +/- 0.17, 1.76 +/- 0.42 for succinic acid, ethanol and acetic acid, respectively. The optimum C:N ratios for the different substrates did not change in response to an increased flow rate. At a C: N ratio of 3:1 and a HRT of 1.5 h, the immobilized cells did not retain their activity. Apart from the difference in the effectivity between the electron donors, the main influence on the denitrification rate was exerted by the flow rate The results of this study demonstrated that Pseudomonas butanovora can utilize all three of these carbon sources to achieve a high rate of denitrification.