Several isolates were obtained from sporocarps of Amanita caesarea (Scop.: Fr.) Pers. associated with Quercus suber and Castanea sativa coming from the southwest of Spain. Culture conditions were optimized for these isolates. The largest radial growth was obtained at pH 6-7, and optimal growth temperature was 24-28 degrees C depending on the isolate. Albumin bovine and nitrate produced the largest patch size diameters, but the greatest mycelium dry weight yields were obtained with ammonium. Mannitol produced the largest radial growth, and mannitol and glucose yielded the biggest mycelium dry weights. Although variations in growth behaviour between isolates were observed, only one internal spacer sequence-restriction fragment length polymorphism type was obtained.