The monoamine neurotransmitter serotonin is released from spinal terminals of nucleus raphe magnus (NRM) neurons and important in sensory and motor control, but its pattern of release has remained unclear. Serotonin was measured by the high-resolution method of fast cyclic voltammetry (2 Hz) with carbon-fiber microelectrodes in lumbar segments (L3-L6) of halothane-anesthetized rats during electrical stimulation of the NRM. Because sites of serotonin release are often histologically remote from membrane transporters and receptors, rapid emergence into aggregate extracellular space was expected. Increased monoamine oxidation currents were found in 94% of trials of 50-Hz, 20-s NRM stimulation across all laminae. The estimated peak serotonin concentration averaged 37.8 nM (maximum 287 nM), and was greater in dorsal and ventral laminae (I-III and VIII-IX) than in intermediate laminae (IV-VI). When measured near NRM-evoked changes, basal monoamine levels (relative to dorsal white matter) were highest in intermediate laminae, while changes in norepinephrine level produced by locus ceruleus (LC) stimulation were lowest in laminae II/III and VII. The NRM-evoked monoamine peak was linearly proportional to stimulus frequency (10-100 Hz). The peak often occurred before the stimulus ended (mean 15.6 s at 50 Hz, range 4-35 s) regardless of frequency, suggesting that release per impulse was constant during the rise but fell later. The latency from stimulus onset to electrochemical signal detection (mean 4.2 s, range 1-23 s) was inversely correlated with peak amplitude and directly correlated with time-to-peak. Quantitative modeling suggested that shorter latencies mostly reflected the time below detection threshold (5-10 nM), so that extrasynaptic serotonin was significantly elevated well within 1 s. Longer latencies (>5 s), which were confined to intermediate laminae, appeared mainly to be due to diffusion from distant sources. In conclusion, except possibly in intermediate laminae, serotonergic volume transmission is a significant mode of spinal control by the NRM.