Sequences of at least three new families of retrotransposons (Tto1-Tto3) were amplified by PCR from cDNA prepared from protoplasts of an established tobacco cell line, based on the fact that certain amino acids are highly conserved in the reverse transcriptases encoded by retrotransposons. Structural analysis indicates that Tto1 is 5.5 kb long and has features typical of retrotransposons. Transcription of Tto1 starting in the long terminal repeat was active only in cultured cells. Protoplast formation enhanced the transcription. The copy number of Tto1 increased 10-fold in established cell lines; it also increased in plants regenerated from tissue cultures and in transgenic plants. These results indicate that Tto1 is activated during tissue culture. This is the first demonstration of activation of a plant retrotransposon by tissue culture. The copy number of Tto2 and a previously isolated transposon, Tnt1, also increased in established cell lines, indicating that these two retrotransposons may also be activated by tissue culture. These three retrotransposons are cryptic in normally propagated plants: no difference in the copy number was observed between individuals of the same cultivars or even between different cultivars.