The structure of G-bands in human metaphase chromosomes was analyzed by comparison between light microscopic and atomic force microscopic (AFM) images of the same chromosomes. G-bands of the chromosomes were made by trypsin treatment followed by staining with a Giemsa solution. The banded chromosomes examined by light microscopy were dried either in air or in a critical point-drier, and observed by non-contact mode AFM. Air-dried chromosomes after G-band staining showed alternating ridges and grooves on their surface, which corresponded to light-microscopically determined G-positive and G-negative bands, respectively. At high magnification, the G-positive ridges were composed of densely packed chromatin fibers, while the fibers were loose in the G-negative grooves. Fibers bridging the gap between sister chromatids of a mitotic pair were often found, especially in the G-positive portions. These findings suggest that the G-banding pattern reflects the high-order structure of human metaphase chromosomes.