Use of specific stains permits analysis of the frequency of nucleolus-associated heterochromatin in chromosomes 1 and 9 from human fibroblasts. In 81 per cent of interphase nuclei the heterochromatic segment of both No. 1 chromosomes is associated with the nucleolus, while in 19 per cent only one heterochromatic segment shows such an association with the other occupying a random position in the nucleoplasm. The nucleolar association of chromosome 9 heterochromatin is less constant: in 42.3 per cent of the nuclei both segments are associated with the nucleolus, in 39 per cent of the nuclei only one heterochromatic segment presents such an association, and in 18.7 per cent neither of the two heterochromatic segments is in nucleolar association. In 6 per cent of the cells, one or two chromosome 9 heterochromatic segments are in contact with the nuclear membrane. In situ hybridization using tritium-labeled 28S and 18S RNA shows that in the interphase nucleus the acrocentric short arms, carriers of ribosomal cistrons, are associated with the nucleolus. These observations demonstrate the complexity of the nucleolus-associated chromatin which, in addition to segments of chromosomes 1, 9, 13, 14, 15, 21, 22, may include the Y chromosome. They also confirm that the nucleolus constitutes one of the orientation points determining the relative localization of chromosomes in the interphase nucleus.