Application of neuroactive substances, including monoamines, is common in studies examining the spinal mechanisms of sensation and behavior. However, affected regions and time-courses of transmitter activity are uncertain. We measured the spatial and temporal distribution of serotonin (5-HT) in the lumbosacral spinal cord of halothane-anesthetized adult rats, following its intraspinal microinjection or surface application. Carbon fiber microelectrodes (CFMEs) were positioned at various locations in the spinal cord and oxidation currents corresponding to extracellular 5-HT were measured by fast cyclic voltammetry. Intraspinal microinjection of 5-HT (100 µM, 1-3 µl) produced responses that were most pronounced at CFMEs positioned &#8804; 800 µm from the drug micropipette: 5-HT concentration was significantly higher (1.43 % vs. less than 0.28% of initial concentration) and response latency was shorter (67.1 vs 598.2 s) compared to more distantly positioned CFMEs. Treatment with the selective 5-HT reuptake inhibitor clomipramine only slightly affected the spread of microinjected 5-HT. Surface application over several segments led to a transient rise in concentration that was usually apparent within 30 seconds and was dramatically attenuated with increasing depth: 0.25% of initial concentration (1 mM) within 400 µm of the dorsal surface and < 0.001% between 1170 and 2000 µm. This initial response to superfusion was sometimes followed by a gradual increase to a new concentration plateau. In sum, compared to bath application, microinjection can deliver about tenfold higher transmitter concentrations, but to much more restricted areas of the spinal cord.