Abstract HeLa mitochondrial 4 s RNA has been covalently coupled to the electron opaque label, ferritin, which is visible in the electron microscope. Mixtures of HeLa mitochondrial 12 s ribosomal RNA, 16 s rRNA and/or the 4 s RNA-ferritin conjugate have been hybridized to separated heavy (H) and light (L) strands of HeLa mitochondrial DNA, or to a mixture of H and L strands. The relative positions of the duplex regions corresponding to the 12 s and 16 s rRNA—DNA hybrids and of the ferritin-labeled 4 s RNA's have been mapped in the electron microscope after spreading the DNA strands by the formamide modification of the basic protein film technique. The 12 s and 16 s duplex regions have lengths of 0·-26 ± 0.04 μm and 0.46 ± 0.07 μm, respectively. They are separated by a single-strand region of length 0.047 ± 0.017 μm, corresponding to 160 ± 60 nucleotides. There are nine reproducible binding sites for 4 s RNA on the H strand. One such site lies within the spacer region between the 12 s and 16 s coding sequences, one site is immediately adjacent to the other side of the 12 s sequence and one is adjacent to the other side of the 16 s sequence. The other 4 s sites are rather evenly spaced along the DNA strand of total length 15,600 nucleotides, except that two of them are clustered with a spacing of 120 ± 30 nucleotides between them. There are three 4 s RNA coding sequences on the L strand, separated from one another by 2280 and 3900 nucleotides, respectively.