Transgenic plastids offer unique advantages in plant biotechnology, including high-level foreign protein expression and reducing environmental contamination risks. Here, the development of a specific plastid vector for lettuce, (Lactuca sativa L. TN-96-39), was described using of rbcl and accD as target fragments, Prrn promoter, psbA terminator and a gene cassette for antibiotic resistance. The species-specific vector for plastid transformation to Lactuca sativa, called pCL 96-39 (TN-96-39 (Iranian line) chloroplast plasmid), has been sequenced. Plastid transformation was optimized by biolistic bombardment. Transfomants were confirmed by PCR and also histochemical test for GUS protein production. Beta-glucoronidase production was confirmed by SDS-PAGE electrophoresis in transformants. This system will open up new possibilities for the efficient production of edible vaccines, pharmaceuticals, and antibodies in plants.