The hypaphorine concentration in Pisolithus tinctorius Coker & Couch hyphae colonizing Eucalyptus roots was 3 to 5 times higher than in adjacent parts of the fungal colony. This phenomenon, observed 24 h after inoculation, was also recorded in several-month-old, well-established ectomycorrhizas. Accumulation was controlled by specific root-derived diffusible molecules: it can be induced through a membrane, but not by non-host plants. In pure culture, high hypaphorine concentration was found only in the youngest mycelium, i.e. the outer 2 mm of the colony. Fungal hypaphorine had no IAA-like activity on Eucalyptus root development and therefore could not be considered as an auxin analogue; instead, a strong reduction of root hair elongation was recorded.