1. The Rumen Simulation Technique (Rusitec) was used in a series of long-term experiments to study the distribution and changes of urease (EC 220.127.116.11) activity in a heterogeneous fermentation system.
2. It was shown that in Rusitec the high urease activity from the inoculum decreased to low values, that the rate of decrease was consistent with simple dilution of ureolytic micro-organisms and that the urease activity could be restored to original values by infusion of urea into the reaction vessels. The magnitude of this urease activity was a direct function of the amounts of urea infused. Single daily additions of the same or greater amounts of urea in food or as solid failed to increase the urease activity significantly.
3. In general, urease activity increased 2–6 h after feeding and the increases were greater with roughage diets.
4. The ureolytic activity per unit volume was always higher in compartment 2 (space occupied by micro-organisms that are loosely associated with the solid) than in compartment 1 (strained rumen contents) or compartment 3 (space occupied by microbial population that cannot be washed out of the solid matrix).
5. The distribution of urease activity between the compartments was different from the distribution of certain other enzymes (e.g. protease and alkaline phosphatase (EC 18.104.22.168)).
6. Apart from the boundary region, the concentrations of urease, ammonia and volatile fatty acids in compartment 2 were constant, while the concentrations of protein, DNA and another enzyme (alkaline phosphatase) increased with the depth of the compartment. Specific urease activity (per unit weight of protein or DNA) was much higher in compartment 1 than in compartment 2 and it decreased markedly with depth of compartment.
7. The concentrations of ammonia were always much higher in the solid matrix (compartments 2 and 3) than in the free suspension of micro-organisms (compartment 1). There was a linear relation between these two quantities.
8. The results are discussed in relation to published work on the entry and metabolism of urea in the rumen.