The quantitative contribution of fatty acids and CO(2) to methanogenesis was studied by using stirred, 3-liter bench-top digestors fed on a semicontinuous basis with cattle waste. The fermentations were carried out at 40 and 60 degrees C under identical loading conditions (6 g of volatile solids per liter of reactor volume per day, 10-day retention time). In the thermophilic digestor, acetate turnover increased from a prefeeding level of 16 muM/min to a peak (49 muM/min) 1 h after feeding and then gradually decreased. Acetate turnover in the mesophilic digestor increased from 15 to 40 muM/min. Propionate turnover ranged from 2 to 5.2 and 1.5 to 4.5 muM/min in the thermophilic and mesophilic digestors, respectively. Butyrate turnover (0.7 to 1.2 muM/min) was similar in both digestors. The proportion of CH(4) produced via the methyl group of acetate varied with time after feeding and ranged from 72 to 75% in the mesophilic digestor and 75 to 86% in the thermophilic digestor. The contribution from CO(2) reduction was 24 to 29% and 19 to 27%, respectively. Propionate and butyrate turnover accounted for 20% of the total CH(4) produced. Acetate synthesis from CO(2) was greatest shortly after feeding and was higher in the thermophilic digestor (0.5 to 2.4 muM/min) than the mesophilic digestor (0.3 to 0.5 muM/min). Counts of fatty acid-degrading bacteria were related to their turnover activity.