Lactuca sativa is widely used as a leafy vegetable belonging to the Asteraceae family. Cotyledonary leaves of in vitro lettuce seedlings were transformed by plasmids containing the GCHI gene. Also the effects of number of days of pre-cultivation and co-cultivation of explants were examined on the transformation frequency. After co-cultivation, the explants were transferred onto the regeneration medium containing 200 mg/l ceftriaxone and 20 mg/l kanamycin. The best callogenesis was obtained in the MS medium containing 0.05 mg/l NAA and 0.4 mg/l BAP. Shoot regeneration was achieved after transferring the calli onto the MS medium containing 0.05 mg/l NAA and 0.4 mg/l BAP. Finally, the shoots were rooted on the MS medium without growth regulators. A PCR analysis of the genomic DNA confirmed the presence of GCHI genes in both T0 and T1 plants. PCR analysis of T0 plants revealed that 10 out of 52 plants were positive for the GCHI gene. The transformation frequency was 19%.