The in vivo association with proteins of HeLa cell mitochondrial DNA (mtDNA) has been investigated by analyzing the pattern of in situ crosslinking of the DNA by 4'-hydroxymethyl-4, 5',8-trimethylpsoralen (HMT). Either isolated mitochondria or whole cells were irradiated with long wavelength UV light in the presence of ths psoralen derivative, and the mtDNA was then isolated and analyzed in the electron microscope under totally denaturing conditions. No evidence of nucleosomal structure was found. The great majority of the molecules (approximately 90%) had a double-stranded DNA appearance over most of their contour length, with one to several bubbles occupying the rest of the contour, while the remaining 10% of the molecules appeared to be double-stranded over their entire length. Analysis of restriction fragments indicated the presence, in approximately 80% of the molecules, of a protected segment (300 to 1500 bp long) in a region which was centered asymmetrically around the origin of replication so as to overlap extensively the D-loop. Control experiments showed that at most 30% of the bubbles found near the origin could represent D-loops or expanded D-loops: furthermore, it could be excluded that some sequence peculiarity would account for the preferential location of bubbles near the origin of replication. The data have been interpreted to indicate that, in at least 55% of HeLa cell mtDNA molecules, the region around the origin is protected from in situ psoralen crosslinking by proteins or protein complexes which are associated in vivo with the DNA.