To demonstrate three-dimensionally the process of the collagen fibril growth, the bottom of culture dishes with human osteosarcoma cells (NOS-1) and their extracts were examined by conventional scanning electron microscopy (SEM). Backscattered electron (BSE) imaging of SEM was also applied to the specimens, which were stained with phosphotungustic acid and uranyl acetate. Conventional SEM images showed several stages of collagen fibril assembly. Short collagen fibrils with tapered ends were distributed at the bottom of the dish just beneath and/or around the cultured cells; they were 1 microm long and 20-30 nm in diameter at the thickest middle portion. These fibrils were often twisted and united in a right helical direction, and consequently increased in length (5-10 microm) and diameter (more than 100 nm). In BSE images, the periodical bands stained with phosphotungstic acid and uranyl acetate were visualized throughout the individual fibrils. The banding pattern indicated that the polarity of the collagen molecules was unidirectional; namely, that all molecules were pointed in the same direction throughout the length of the fibrils.