Copper (Cu) is the main co-factor in the functioning of the ammonia monooxygenase (AMO) enzyme, which is responsible for the first step of ammonia oxidation. We report a greenhouse-based pot experiment that examines the response of ammonia-oxidizing bacteria and archaea (AOB and AOA) to different bioavailable Cu concentrations in three pastoral soils (Recent, Pallic, and Pumice soils) planted with ryegrass (Lolium perenne L.). Five treatments were used: control (no urine and Cu), urine only at 300 mg N kg-1 soil (Cu0), urine + 1 mg Cu kg-1 soil (Cu1), urine + 10 mg Cu kg-1 soil (Cu10), and urine + 100 mg Cu kg-1 soil (Cu100). Pots were destructively sampled at Day 0, 1, 7, 15, and 25 after urine application. The AOB/AOA amoA gene abundance was analyzed by real-time quantitative polymerase chain reaction at Days 1 and 15. The AOB amoA gene abundance increased 10.0- and 22.6-fold in the Recent soil and 2.1- and 2.5-fold in the Pallic soil for the Cu10 compared with Cu0 on Days 1 and 15, respectively. In contrast, the Cu100 was associated with a reduction in AOB amoA gene abundance in the Recent and Pallic soils but not in the Pumice soil. This may be due to the influence of soil cation exchange capacity differences on the bioavailable Cu. Bioavailable Cu in the Recent and Pallic soils influenced nitrification and AOB amoA gene abundance, as evidenced by the strong positive correlation between bioavailable Cu, nitrification, and AOB amoA. However, bioavailable Cu did not influence the nitrification and AOA amoA gene abundance increase.