Abstract: The in vivo rat mydriasis model provides more information on ligand-receptor interactions than in vitro studies. It allows assessment which receptors (α2-adrenergic or imidazoline) participate in mydriatic effect produced by imidazoline compounds, regarding the agonistic and antagonistic properties and the strength of the reference ligands action. The reference imidazoline agents can be tested within a wide range of doses, in vivo in the animals. The experiments were performed to assess the mydriatic effects of clonidine and rilmenidine with and without pretreatment of rats with yohimbine, a reference α2-adrenoceptor antagonist. Additionally, compound AGN 192403, regarded as a selective antagonist of I1-imidazoline receptors was used in experiments. However, the previous data indicated that AGN 192403 was devoid of both agonist and antagonist activity in some functional assays. As a result of further investigation, AGN 192403 has been reported as an antagonist to bind selectively at the I1 imidazoline binding site. The α2-adrenomimetic potency of clonidine appeared to be stronger than rilmenidine. Maximum mydriatic effect for clonidine and rilmenidine was found at doses of 30 and 1000 μg kg-1, respectively. Yohimbine administration caused parallel shift of the dose-effect curve for both rilmenidine and clonidine to the right. Preliminary experiments with AGN 192403 seem to confirm the hypothesis that the involvement of I1-imidazoline receptors in mydriatic effects of imidazolines is marginal, if any. The present study shows that rilmenidine has about 30 times weaker affinity to central α2-adrenoceptors than clonidine. Yohimbine inhibits pupil dilation evoked by rilmenidine and clonidine. That confirms the decisive role of α2-adrenoceptors in mydriatic effect and suitability of the Rat Pupil Mydriasis Model for studies of receptor selectivity of the centrally acting drugs.