The mechanism of C-banding was analyzed on the basis of the structural changes of the 30 nm chromatin fibre using scanning electron microscopy (SEM). SEM of non-banded metaphase spreads of L-cells revealed chromosomes consisting of 30 nm chromatin fibre loops along the entire length. No marked difference in both the dimension and appearance of such looped structures was discernible between the centromeric region and the rest of the chromosome. In contrast, C-banded chromosomes exhibited a conspicuous alteration of the fibre conformation in the centromeric region. The looped, fibrous structures were almost completely lost from this region, while the non-centromeric region still exhibited fibrous structures with slightly different appearances compared with those observed in the control chromosomes. On the other hand, results obtained using fluorescence microscopy showed that more DNA retained in the centromeric region than in the non-centromeric region. Since the analytical experiments exhibited that the characteristic collapsed state of the centromeric region occurred only with the alkali treatment but neither with the 2 x SSC nor acid treatments, the centromeric heterochromatin seemed to contain some specific protein which should be sensitive to alkali. The structurally collapsed but subsequently compact centromeric region may become more, or still, resistant to the DNA extraction due to the 2 x SSC treatment and the centromeric chromatin thus retained may be visualized as the C-band.