Transposable elements can be identified by their ability to induce mutant alleles at new loci. The retrotransposon family is thought to transpose through an RNA intermediate and has many similarities to vertebrate proretroviruses. In plants, retrotransposons have been described in maize, Arabidopsis and wheat, and non-viral retroposons in maize. Most of these elements, however, have been found as non-mobile integrated units. Here, we report the isolation of the first tobacco (Nicotiana tabacum) transposable element, Tnt1, which seems to be the most complete mobile retrotransposon characterized in higher plants. Tnt1 has been isolated after its transposition into the nitrate reductase (NR) structural gene of tobacco, and transposition events have been detected through in vitro selection of spontaneous NR-deficient (NR-) mutant lines in cell cultures derived from tobacco mesophyll protoplasts. Tnt1 is 5,334 nucleotides long, contains two 610-base-pair-long terminal repeats and a single open reading frame of 3,984 nucleotides. Comparison of the Tnt1 open reading frame coding potential with those of the Drosophila melanogaster copia retrotransposon, yeast Ty retrotransposon, and vertebrate proretroviruses revealed that Tnt1 is closely related to copia and carries all the functions known to be required for autonomous transposition by reverse transcription.