Marker-free transgenic tomato plants harboring a synthetic Bacillus thuringiensis endotoxin gene, cryIAc, were obtained by using a chemically regulated, Cre/loxP-mediated site-specific DNA recombination system, in which the selectable marker neomycin phosphotransferase gene flanked by two directly oriented loxP sites was located between the cauliflower mosaic virus 35S promoter and a promoterless cryIAc. Upon induction by 2 microM beta-estradiol, sequences encoding the selectable marker and cre sandwiched by two loxP sites were excised from the tomato genome, leading to activation of the downstream endotoxin gene cryIAc with high expression levels as shown by Northern blot and ELISA assay (250-790 ng g(-1) fresh wt) in T(1) generation. For transgenic line with single transgenic loci, 15% of T(1) progenies were revealed marker-free. This autoexcision strategy provides an effective approach to eliminate a selectable marker gene from transgenic tomato, thus expediting the public acceptance of genetically modified crop.