Yellow leaf curl disease of tomato caused by tomato yellow leaf curl virus (TYLCV: a begomovirus) causes severe damage. In general, the virus is transmitted by the whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) and has been assumed not to be mechanically transmissible. Because mechanical inoculation has great practical uses, e.g., screening for resistant tomato varieties, here we developed a method using a toothbrush to inoculate plants of susceptible tomato cv. House-Momotaro with TYLCV. The inoculum solution (buffer ingredients, molarity, pH, dilution of viruliferous leaf sap) were optimized to achieve the highest infection rates. After 35 days, characteristic symptoms appeared on the inoculated tomato plants, and infection of TYLCV was confirmed in non-inoculated upper leaves by polymerase chain reaction. The highest infection rate (73.3%) was obtained using 100 mM potassium phosphate buffer (pH 7.0) containing 0.1% (v/v) 2-mercaptoethanol at a 1000-fold volume of macerated infected leaf tissue. TYLCV was transmitted mechanically at 73.3 % infectivity in proportion of plants inoculated.