In time‐course experiments, the amounts of phytoalexin in hypocotyls of the resistant bean cultivar Flor de Mayo were determined after infection with Colletotrichum lindemuthianum. Phaseollin and phaseollidin accumulated as a defence response. However, phaseollidin accumulated earlier than phaseollin and at greater concentrations (155 + 14 compared with 120 + 9 μg g−1 fresh weight). Phaseollidin was found conjugated as a glucoside in the tissue. Fungal treatment apparently led to a decline of approximately 50% in phaseollidin conjugate after only 7 h after infection. Isolation of vacuoles confirmed the presence of phaseollidin glucosides in this organelle. Treatment of the tissue or isolated protoplasts with a fungal elicitor also produced a decrease, by half, of phaseollidin conjugate concentrations from vacuoles isolated from both sources. The contribution of pre‐existing pools of phaseollidin glycoside to the accumulation of this phytoalexin is discussed.