Adventitious roots (ARs) are post-embryonic roots formed in planta by tissues of the primary root in secondary vascular structure and by tissues of the aerial organs. Indole-3-acetic acid (IAA), and its natural precursor indole-3-butyric acid (IBA), control AR formation in planta and in vitro, however IBA roles have to be elucidated. Arabidopsis thin cell layers (TCL) consist of stem inflorescence tissue external to the vascular system and 10 microM IBA applied with 0.1microM Kinetin induce AR formation from stem TCL. In the Arabidopsis transversal stem cuttings, it has been hypothesized that the induction of AR formation by exogenous IBA occurs by an interaction with the endogenous IAA content, but there is no information about the interaction between the two auxins in the TCLs. In Arabidopsis seedlings it has been demonstrated that IBA is sufficient to stimulate IAA transport because PIN-FORMED1 (PIN1) IAA-efflux carrier, AUXIN RESISTANT1 (AUX1) and LIKE AUXIN RESISTANT3 (LAX3) IAA-influx carriers are active also in the presence of IBA alone. The WEAK ETHYLENE-INSENSITIVE2/ANTHRANILATE SYNTHASE alpha1 (WEI2/ASA1) and WEI7/ANTHRANILATE SYNTHASE beta1 (ASB1), are genes involved in IAA-biosynthesis and required for AR formation in Arabidopsis seedlings. It is unknown whether the same genes are involved in AR-formation by TCLs. The aim of the research was to determine the endogenous levels of IBA and IAA at the onset of the culture in Arabidopsis TCLs. Another aim was to understand whether IBA alone was able to induce AR formation in TCL, whether the IAA transport by PIN1, LAX3, and AUX1 was affected, whether an IBA conversion into IAA was needed, and whether an IAA biosynthesis by WEI2/ASA1 and WEI7/ASB1 was also involved. Results indicate that IBA induced AR-formation by conversion into IAA, with this process involving nitric oxide formation and activity, and by positively affecting IAA-transport and ASA1/ASB1-mediated IAA-biosynthesis.