The actions of 5‐methoxytryptamine (5‐MeOT), N,N‐dimethyltryptamine (DMT), 5‐hydroxy‐N,N‐dimethyltryptamine (bufotenine, 5‐HODMT) and 5‐methoxy‐N,N‐dimethyltryptamine (5‐MeODMT), and their interactions with 5‐hydroxytryptamine (5‐HT), acetylcholine, (–)‐noradrenaline, and glutamate were studied by microiontophoresis on single neurones in the brain stem of rats anaesthetized with urethane or decerebrate cats. Like d‐lysergic acid diethylamide (LSD 25) the three psychotomimetic derivatives (DMT, 5‐HODMT, 5‐MeODMT) specifically antagonized 5‐HT excitations of single neurones, but the non‐psychotomimetic 5‐MeOT had no antagonistic effects. In contrast to LSD 25, the psychotomimetic tryptamines only rarely antagonized glutamate effects, indicating that the excitatory 5‐HT receptors and the glutamate receptors on the same neurones may be closely related spatially, but are separate. The methylated tryptamine derivatives were able to mimic the actions of 5‐HT on neurones. The non‐psychotomimetic 5‐MeOT was most potent in this respect, while the other three derivatives which are psychotomimetic, were less active. The 5‐HT mimicking actions of 5‐MeOT were the same in rats pretreated with p‐chlorophenylalanine or reserpine as in untreated rats. It therefore seems that the 5‐HT mimicking actions are unlikely to be due to release of 5‐HT, but are due to direct actions on 5‐HT receptors. The evidence presented supports the hypothesis that LSD‐like psychotomimetics act by an antagonism of 5‐HT in the lower brain stem, and is not compatible with the suggestion that the psychotomimetic action of these drugs is related to 5‐HT receptor stimulation.