The control of replication can facilitate a viral amplicon to reach high expression levels by enabling the virus to escape host defence mechanisms and reducing the deleterious effects of viral infection. We have developed a novel system to regulate amplicon expression by controlling the temperature of plant growth. Nicotiana benthamiana plants were transformed at two different temperatures with a cDNA copy of the Plum pox potyvirus genome harbouring the open reading frame 2 of Porcine circovirus 2 between the nuclear inclusion protein b and coat protein coding sequences. Although transformation at 27 degrees C mainly yielded nonexpressing amplicons, lines with a tight control of amplicon expression were obtained. Viral replication was not detected in these plants when germinated at 28 degrees C, but was observed when the plants were shifted to 20 degrees C. In lines transformed at 24 degrees C, although the amplicon was expressed at 28 degrees C, viral accumulation was low and caused only minor growing defects. Viral replication was enhanced in these plants by shifting the temperature to 20 degrees C; under such conditions, the amplicon reached higher and more persistent expression levels than in plants transformed at 27 degrees C. These results demonstrate the utility of temperature regulation to control viral amplicon expression.