Fischer 344 (F344) and Lewis rat strains have been shown to exhibit different vulnerability to development or maintenance of opioid seeking behaviours probably due to differences in the endogenous opioid system. Since opioid and α2-adrenergic mechanisms closely interact in nociception and substance abuse, strain differences may be expected to affect α2-adrenoceptor-mediated events. The sensitivity of these two strains to α2-adrenoceptor-mediated antinociception has been reported to be markedly different. In this work we have further studied the function of α2-adrenoceptors in F344 and Lewis rats by means of several in vivo and in vitro procedures. Comparative studies of [3H]RX821002 and [35S]GTPγS binding revealed that α2-adrenoceptors could be slightly more responsive to agonist stimulation in the brain cortex of F344 rats, which is in agreement with previous antinociception studies. However, these differences were modest, not observed in the spinal cord and did not translate into functional differences concerning the effects of clonidine on vas deferens contractility and body temperature. Conditioning experiments showed that a moderate dose of clonidine, which is relevant in antinociceptive and opioid antiwithdrawal studies, induces a robust place aversion which is also equivalent in F344 and Lewis rats. This finding underlies the consistency of the effect and its independency of genetic differences between both rat strains. It seems therefore that the pharmacological properties of α2-adrenoceptors are similar in F344 and Lewis rats, and thus the previously reported differences in clonidine-induced antinociception could be attributed to other factors such as dissimilar endogenous function of specific noradrenergic pathways.