Inhibition of the fermentation of propionate to methane and carbon dioxide by hydrogen, acetate, and propionate was analyzed with a mesophilic propionate-acclimatized sludge that consisted of numerous flocs (size, 150 to 300 mum). The acclimatized sludge could convert propionate to methane and carbon dioxide stoichiometrically without accumulating hydrogen and acetate in a propionate-minimal medium. Inhibition of propionate utilization by propionate could be analyzed by a second-order substrate inhibition model (shown below) given that the substrate saturation constant, K(s), was 15.9 muM; the substrate inhibition constant, K(i), was 0.79 mM; and the maximum specific rate of propionate utilization, q(m), was 2.15 mmol/g of mixed-liquor volatile suspended solids (MLVSS) per day: q(s) = q(m)S/[K(s) + S + (S/K(i))], where q(s) is the specific rate of propionate utilization and S is the initial concentration of undissociated propionic acid. For inhibition by hydrogen and acetate to propionate utilization, a noncompetitive product inhibition model was used: q(s) = q(m)/[1 + (P/K(p))], where P is the initial concentration of hydrogen or undissociated acetic acid and K(p) is the inhibition constant. Kinetic analysis gave, for hydrogen inhibition, K(p(H(2))) = 0.11 atm (= 11.1 kPa, 71.5 muM), q(m) = 2.40 mmol/g of MLVSS per day, and n = 1.51 and, for acetate inhibition, K(p(HAc)) = 48.6 muM, q(m) = 1.85 mmol/g of MLVSS per day, and n = 0.96. It could be concluded that the increase in undissociated propionic acid concentration was a key factor in inhibition of propionate utilization and that hydrogen and acetate cooperatively inhibited propionate degradation, suggesting that hydrogenotrophic and acetoclastic methanogens might play an important role in enhancing propionate degradation to methane and carbon dioxide.