Abstract: Rice chloroplast 16S promoter was isolated by PCR from rice chloroplast genomic DNA. In order to enhance the ability of translation, SD sequence from rbcL gene was introduced into the downstream of the promoter. The sequence analysis showed that the isolated fragment and the rice（Oryza sativa） chloroplast 16S promoter shared 100% homology except the introduced SD sequence. A Nicotiana tabacum chloroplast transformation vector pR16S has been constructed. It contains not only the chimeric gene bar and gfp gene regulated by 16S promoter and psbA teminator, but also two homologous fragments, trnH-psbAand trnK. The plasmid pR16S has been transferred to Nicotiana tabacum by biolistic method. The transformants were identified by Southern-blotting，Northern-blotting and genetic analysis of progenies. The results showed that 16S promoter worked effectively and the foreign gene had been integrated in the chloroplast genome and inherited maternally to the progenies.