By using a novel fractionation procedure we show that the chloroplast chromosome is specifically bound to the spinach thylakoid in the region of the inverted repeat, near the 16S and 23S rRNA genes. Central to the method is the use of restriction endonucleases, followed by ethylenediaminetetraacetic acid (EDTA) treatment to loosen or uncoil the nucleoid by the removal of specific proteins. The data we present provide a basis for understanding the molecular mechanism of membrane-bound cpDNA function.