The 2,5-dimethoxyphenethylamine (2, 5-PEA) scaffold is recognized as a motif conferring potent agonist activity at the seroto-nin 2A receptor (5-HT2AR). The 2,5-dimethoxy motif is present in several classical phenethylamine psychedelics such as mesca-line, TMA-2, DOM, DOI, DOB, 2C-B and 2C-I, and it has previously been suggested that this structural motif is essential for 5-HT2AR activation. In the present study we present data that challenges this assumption. The 2- and 5-desmethoxy derivatives of 2C-B and DOB were synthesized and their pharmacological profiles evaluated in vitro at 5-HT2AR and 5-HT2CR in binding and functional assays and in vivo by assessing their induction of Head Twitch Response in mice. Elimination of either the 2- or 5-methoxy leads to a modest drop in binding affinity and functional potency at 5-HT2AR and 5-HT2CR, which was more pro-nounced upon removal of the 5-methoxy. However, this trend was not mirrored in vivo, as removal of either methoxy group resulted in significant reduction in the compounds ability to induce the Head Twitch Response in mice. Thus, the 2,5-dimethoxyphenethylamine motif appears to be important for in vivo potency of phenethylamine 5-HT2AR agonists, but this does not correlate to the relative affinity and potency of the ligands at the recombinant 5-HT2AR.