This study was designed to control plant fertility by cell lethal gene Barnase expressing at specific developmental stage and in specific tissue of male organ under the control of Crellox system, for heterosis breeding of chili pepper (Capsicum annuum L.). Chili pepper inbred lines (A, D, E, and I) were transformed with Cre gene and Barnase gene situated between loxp, separately, by means of Agrobacterium co-culture. In this study, we had established a high transformation system by extensive study of affecting factors including genotype, selection of marker, and lethal dose. Cotyledon with petiole from 9-11-day-old seeding was pre-cultured on media MR [MB (MS mineral + vitamine B5)+BA (6-Benzyladenine) 5.0 mg L(-1)+IAA (indoleacetic acid) 1.0 mg L(-1)+GA(3) (gibberellic acid) 1.0 mg L(-1)+sucrose 3%+agar 6.5 g L(-1)] for 2 d. The explants were infected by Agrobacterium tumefaciens when their OD(600) (optical density at 600 nm) reached 0.6-0.9. After co-cultured for 4-5 d on media MC [MB + BA 5.0 mg L(-1)+IAA 1.0 mg L(-1)+GA(3) 1.0 mg L(-1)+sucrose 3%+agar 6.5 g L(-1)+AS (acetosyringone) 200 mu mol L(-1)], these cotyledons with petiole were cultured on selective differentiation medium in the media MT [MB medium supplemented with BA [5.0 mg L(-1)+IAA 1.0 mg L(-1)+GA(3) 1.0 mg L(-1)+AgNO(3) 5.0 mg L(-1)+CW (coconut water) 5% + Km (kanamycin) 65 mg L(-1)+Cb (carbenicillin) 500 mg L(-1)+3% sucrose + agar 6.5 g L(-1)]. The Kmr (kanamycin resistant) bud rosettes were elongated on selective elongation medium and rooted on rooting medium. PCR and Southern blotting analysis of Kmr plantlet indicated that the foreign genes had been integrated into the genome of pepper. The transgenic plants with Cre gene developed well, blossomed out, and set fruit normally. The transgenic plants with Barnase gene grew well with normal appearance of flower, but they showed different fertility from complete sterility, partial sterility to complete fertility, and similar results were obtained from in vitro pollen germination experiments.