Psychedelic substances such as lysergic acid diethylamide (LSD) and psilocybin show potential for the treatment of various neuropsychiatric disorders^1,2,3. These compounds are thought to mediate their hallucinogenic and therapeutic effects through the serotonin (5-hydroxytryptamine (5-HT)) receptor 5-HT_2A (ref. ⁴). However, 5-HT_1A also plays a part in the behavioural effects of tryptamine hallucinogens⁵, particularly 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT), a psychedelic found in the toxin of Colorado River toads⁶. Although 5-HT_1A is a validated therapeutic target^7,8, little is known about how psychedelics engage 5-HT_1A and which effects are mediated by this receptor. Here we map the molecular underpinnings of 5-MeO-DMT pharmacology through five cryogenic electron microscopy (cryo-EM) structures of 5-HT_1A, systematic medicinal chemistry, receptor mutagenesis and mouse behaviour. Structure–activity relationship analyses of 5-methoxytryptamines at both 5-HT_1A and 5-HT_2A enable the characterization of molecular determinants of 5-HT_1A signalling potency, efficacy and selectivity. Moreover, we contrast the structural interactions and in vitro pharmacology of 5-MeO-DMT and analogues to the pan-serotonergic agonist LSD and clinically used 5-HT_1A agonists. We show that a 5-HT_1A-selective 5-MeO-DMT analogue is devoid of hallucinogenic-like effects while retaining anxiolytic-like and antidepressant-like activity in socially defeated animals. Our studies uncover molecular aspects of 5-HT_1A-targeted psychedelics and therapeutics, which may facilitate the future development of new medications for neuropsychiatric disorders.