The plant hormone ethylene has long been implicated in the process of adventitious root formation (ARF), but the exact details of its action, especially in relation to crosstalk with other environmentally-mediated hormonal signals is still limited. We have identified a potential role for ethylene in ARF from cuttings in woody species in both recalcitrant Populus × canescens (P. alba × P. tremula) and easily-rooted Populus × Canadensis (P. nigra × P. deltoids) species using treatment sets including combinations of the ethylene precursor ACC (aminocyclopropane-1-carboxylic acid), IBA, the ethylene perception inhibitor 1-methylcyclopropene (1-MCP) and the auxin-transport inhibitor triiodo-benzoic acid (TIBA). ACC slightly increased rooting percentages in both genotypes. Exogenous IBA application to produced a similar rooting percentage to ACC. Treatment with 1-MCP produced no rooting in either genotype as did TIBA. ACC was unable to recover rooting in conjunction with TIBA and IBA was unable to recover rooting in conjunction with 1-MCP in both genotypes, indicating that sufficient concentrations of both regulators are required for rooting. Callusing remained widely invariant between treatments in both canadensis and P. canescens, apart from in TIBA and ACC+TIBA treatments where it was increased and reduced respectively. Callusing was similar between 1-MCP and 1-MCP+IBA treatments, but greater than that in TIBA+ACC treatments in both genotypes. Temporal ACC treatments in P. Canadensis show increased rooting between 6 and 24 hours after excision and reduced rooting up to 96h with 1-MCP, suggesting promotive role in the induction period of ARF. Treatments in P. canescens show a decrease in callusing as treatments progress to 96 h on application with ACC. The results indicate that ethylene plays different roles relative to local auxin concentration, suggesting complex fine tuning of cell fate for either callus formation or root initiation may be present in poplar species.