The location of proteins on the mitochondrial DNA (mtDNA) of Drosophila melanogaster was investigated by trimethylpsoralen photoreaction of embryos disrupted by gentle homogenization. After photoreaction, the mtDNA was isolated and the pattern of DNA crosslinking was determined by electron microscopy of the DNA under totally denaturing conditions. In contrast to nuclear DNA, which showed periodic crosslinks indicative of a nucleosome structure, most of each mtDNA molecule exhibited uniformly heavy crosslinking. A 10% region of the mtDNA was, however, protected from psoralen crosslinking in a distinctive manner: five uncrosslinked segments were closely clustered in the mtDNA. Four were 394 +/- 13 (SD) base pairs in size, while the fifth measured about 200 base pairs. These protected segments mapped within the A+T-rich region of the mtDNA, extending from the end of the A+T-rich region near the Bg1 II cleavage site to the center of the A+T-rich region. Protection of this part of the mtDNA from crosslinking was interpreted to be the result of association with proteins in the mitochondrion because mtDNA that was deproteinized before the photoreaction was uniformly crosslinked over its entire length. The origin of replication of the mtDNA is also located at the center of the A+T-rich region, which suggests that the protection from the psoralen photoreaction may be due to proteins involved in membrane attachment or replication.