Summary The mode of action of auxin is based on its non‐uniform distribution within tissues and organs. Despite the wide use of several auxin analogues in research and agriculture, little is known about the specificity of different auxin‐related transport and signalling processes towards these compounds. Using seedlings of Arabidopsis thaliana and suspension‐cultured cells of Nicotiana tabacum (BY‐2), the physiological activity of several auxin analogues was investigated, together with their capacity to induce auxin‐dependent gene expression, to inhibit endocytosis and to be transported across the plasma membrane. This study shows that the specificity criteria for different auxin‐related processes vary widely. Notably, the special behaviour of some synthetic auxin analogues suggests that they might be useful tools in investigations of the molecular mechanism of auxin action. Thus, due to their differential stimulatory effects on DR5 expression, indole‐3‐propionic (IPA) and 2,4,5‐trichlorophenoxy acetic (2,4,5‐T) acids can serve in studies of TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALLING F‐BOX (TIR1/AFB)‐mediated auxin signalling, and 5‐fluoroindole‐3‐acetic acid (5‐F‐IAA) can help to discriminate between transcriptional and non‐transcriptional pathways of auxin signalling. The results demonstrate that the major determinants for the auxin‐like physiological potential of a particular compound are very complex and involve its chemical and metabolic stability, its ability to distribute in tissues in a polar manner and its activity towards auxin signalling machinery.