We have designed an innovative construct (pX6-DAO1) combining the chemical inducible Cre-LoxP system and the conditional selectable marker gene dao1 to obtain marker-free transgenic tobacco plants. Nicotiana tabaccum transgenic lines were regenerated on medium with 6 mM d-alanine. The DNA site-specific recombination was controlled by the inducer s-estradiol. Regeneration on medium containing 5 μM s-estradiol and 8 mM d-valine was not obtained. However, leaf disks from all transgenic lines regenerated in the presence of s-estradiol, although only 9.4 % of regenerated buds developed solid marker-free shoots. Partial recombination was found in 71.7 % of buds, and no recombination was detected in only 18.9 % of buds. Nevertheless, when leaf disks from chimeric shoots were cultured in medium with 8 mM d-valine, only marker-free buds regenerated, and no partial recombinants were detected. Similarly, marker-free plants were produced from T2 seeds, obtained from chimeric s-estradiol-induced T1 plants, with 100 % efficiency in selective d-valine medium.