Neurons in the nucleus raphe magnus were recorded extracellularly from barbiturate-anesthetized rats, and were classified by their responses to noxious mechanical stimulation as either pinch-excited, pinch-inhibited or biphasic (inhibited then excited). They were then subjected to iontophoresis of serotonin, some serotonergic agonists and antagonists, acetylcholine, and γ-amino- n-butyric acid. Serotonin reduced the spontaneous firing of most pinch-inhibited cells (79%). Significantly fewer ( P < 0.05) pinch-excited and biphasic cells were inhibited by serotonin (40% and 45%, respectively); in these two cell classes, the observed response was often excitation (30% and 14%), or inhibition for 10–30 s followed by excitation for the next 1–2 min (25% and 36%). Acetylcholine showed a similar, statistically significant distribution of effects ( P < 0.05), inhibiting all pinch-inhibited neurons ( n = 10) but fewer pinch-excited (53%, n = 17) and biphasic neurons (20%, n = 10). Excitation, or excitation then inhibition, was again found frequently among the remaining pinch-excited and biphasic cells. The effect of γ-amino- n-butyric acid was only inhibitory. In all three nociceptive classes, the serotonin-1A agonist buspirone ( n = 15) was inhibitory (87%) and the serotonin-1C/2 antagonist ketanserin ( n = 20) was excitatory (35%). The mixed serotonin-1/2 antagonist methysergide ( n = 10) was inhibitory (50%) or excitatory (40%). 8-Hydroxy-dipropylaminotetralin ( n = 3) was found to increase spontaneous activity (possibly because of partial serotonin-1A agonism), and ±propranolol ( n = 4) to reduce it (possibly through β-adrenoceptor antagonism, not serotonin-1A antagonism). Ketanserin blocked the excitation induced by applied serotonin ( n = 7), and methysergide blocked the inhibition ( n = 9). It is concluded that serotonin is continuously released in the ventral medial medulla under barbiturate anesthesia, but it may not be the primary mediator of nociceptive responses here, since some of them are opposite in direction from serotonergic responses. Behavioral studies show either no effect or analgesia upon application of serotonin to the raphe magnus; this uncertainty may reflect the complex distribution of serotonergic effects among the nociceptive classes.