Biological and serological assays were conducted with overwintered roots of tobacco and red pepper, capsule of tobacco, and several species of weeds in order to check whether those tissue could serve as a natural source of effection of tobacco mosaic virus (TMV) to field tobacco plants in the spring. Also in this study TMV occurrence was surveyed at several different stages of tobacco growth to see if a natural source discussed above has anything to do with actual appearance of TMV at fields. The results are as follows 1) The most critical period for TMV infection was the time when tobacco plants were handled with human hands; in the case of the modified polyethylene film mulching system it was at transplantation and when this modified system was changed to the regular system, and, in the case of the regular polyethylene film mulching system, the time was at transplanting and at primary sucker control by hands. 2) Roots of tobacco and red pepper were found to carry infective TMV even after overwintering in the soil. 3) Out of 38 weed species belonging to 22 families examined, only two species, Solanum nigrum and Physalis alkekengi var. franchetii were shown to be naturally infected with TMV. 4) TMV was isolated from capsule tissue, but not from immature anther of tobacco.