To elucidate the water-conducting pathways in living trees by the dye injection method, suitable sample preparation procedures are needed. We evaluated quantitatively the properties and concentrations of three dyes (acid fuchsin, basic fuchsin and safranin) widely used for this purpose, and determined the optimal conditions required to avoid artifacts after dye injection into the sap stream of Pieris japonica D. Don. Among the dyes tested, an aqueous solution of acid fuchsin at a concentration of 0.1% or more was the most useful for delineating water movement. In non-transpiring stem segments, the vertical movement of acid fuchsin by capillarity and diffusion from the dye injection site was limited. However, acid fuchsin moved rapidly in the horizontal direction by capillarity and diffusion, and most xylem cells were stained within 2 h. A delay of more than 2 h between dye injection and examination of the tissues greatly reduces the precision of the method. Use of the dye injection method without appropriate, well-defined experimental procedures may give rise to misleading information about the functional water-conducting pathway in living trees.