Rosa multiflora 'Matsushima No. 3' is tolerant to rose root rot disease caused by Pythium helicoides Drechsler, and the tolerance has been revealed through restraining germ tube elongation on penetration into root tissue. Phenolic substances are linked to preventing the germ tube from penetrating tissue; the retardants of germ tube elongation may be hydrolyzates of phenolic substances induced by penetration of a diseased germ tube. In this study, phenolic substances were extracted from the roots of tolerant rose 'Matsushima No. 3' and susceptible rose R. 'Nakashima 91', and we examined the retardant activity. The substances which restrained germ tube elongation of P. helicoides were detected in the root of 'Matsushima No. 3', and these exist as bound phenolic substances (glycosides) in the root. These substances show high retardant activity when hydrolyzed and change to free phenolic substances. The difference between the tolerant and susceptible rose appears to be due to the quantitative difference in the retardants contained in the roots. From the results of HPLC analysis of bound phenolic substances, the peak of the substance eluted before or after 10 minutes in 'Matsushima No. 3' was about three fold higher than that of 'Nakashima 91'. An HPLC fraction containing this peak was tested for retardant activity. This fraction showed significantly higher retardant activity, and we infer that the substance contained in this fraction is related to the expression of tolerance of 'Matsushima No. 3'.