We investigated the diurnal differences in the innermost surface of tracheid cell walls at various developmental stages from cambium to mature xylem. Cryptomeria japonica saplings were cultivated in a growth chamber with a light cycle set at 14 h of light and 10 h of darkness. Samples were collected from the saplings during both the light and dark periods. The innermost surface of cell walls was immunogold-labeled with anti-glucomannan or anti-xylan antiserum and was observed by field emission scanning electron microscopy. Diurnal differences in the aspect of the innermost surface of cell walls were seen only in S(2)-layer-forming tracheids; cellulose microfibrils were clearly evident during the light period, and amorphous material containing glucomannans and xylans was prevalent during the dark period. Cellulose microfibrils were present at the primary-wall formation and S(1)-layer-forming stages, and many warts were observed in the mature tracheids, regardless of the time of sampling. The densities of labeled glucomannans on the innermost surface of cell walls in S(1)- and S(2)-forming tracheids and of labeled xylans in S(2)-forming tracheids during the dark period were significantly higher than those during the light period. These results suggest a diurnal periodicity in the supply of cell wall matrix containing hemicellulose to the innermost surface of developing secondary walls.