Poplar shoots raised in vitro were induced to root by incubation on an auxin (NAA) containing medium for 7 h. After 13 days on an auxin-free medium, 97% of the treated shoots had rooted. The introduction of known antiauxins (PCIB, PBA, POAA) into the rooting expression auxin-free medium, after the 7-h induction by NAA, completely (PCIB and PEA) or severely (POAA) inhibited rooting. The exclusion of calcium from the expression auxin free medium reduced the percentage of rooting by about 42%. The inhibition was still higher in the presence of EGTA, a calcium chelator. Lanthanum chloride, a calcium channel blocker, also completely inhibited rooting, when incorporated into the auxin free medium, with or without calcium. These results support previous hypotheses about the implication of both endogenous auxin and calcium in the late phases of the adventitious rooting process.