We have analyzed the stress-induced amplification of the tobacco Tnt1 element, one of the rare active plant retrotransposons. Tnt1 mobility was monitored using the retrotransposon-anchored SSAP strategy that allows the screening of multiple insertion sites of high copy number elements. We have screened for Tnt1 insertion polymorphisms in plants regenerated from mesophyll leaf cells, either via explant culture or via protoplast isolation. The second procedure includes an overnight exposure to fungal extracts known to induce high levels of Tnt1 transcription. Newly transposed Tnt1 copies were detected in nearly 25% of the plants regenerated via protoplast isolation, and in less than 3% of the plants derived from explant culture. These results show that Tnt1 transcription is followed by transposition, and that fungal extracts efficiently activate Tnt1 mobility. Transcription appears to be the key step to controlling Tnt1 amplification, as newly transposed Tnt1 copies show high sequence similarities to the subpopulations of transcribed Tnt1 elements. Our results provide direct evidence that factors of microbial origin are able to induce retrotransposon amplification in plants, and strengthen the hypothesis that stress modulation of transposable elements might play a role in generating host genetic plasticity in response to environmental stresses.