We carried out experiments to investigate the mechanisms of serotonin-induced axonal excitability changes using isolated dorsal columns from young (seven to 11-day-old) Long–Evan's hooded rats. Conducting action potentials were activated by submaximal (50%) and supramaximal constant current electrical stimuli and recorded with glass micropipette electrodes. In experiment 1, to study Ca 2+-mediated mechanisms, we superfused the preparations with Ringer solutions containing varying Ca 2+ concentrations. Following superfusion with Ca 2+-free Ringer solution for 4 h, we tested initial responses to serotonin agonists. Studies then were repeated after preparations had been washed for 1 h with Ringer solution containing 1.5 mM Ca 2+ and 1.5 mM Mg 2+. After 4 h superfusion of Ca 2+-free Ringer solution, quipazine (a serotonin 2A agonist, 100 μM) did not induce significant axonal excitability changes (amplitude change of 1.4±1.3%, percentage of predrug control level, ±S.D., n=6). A 100 μM concentration of 8-hydroxy-dipropylaminotetralin (a serotonin 1A agonist) reduced response amplitudes by 36.3±4.2% (±S.D., P<0.0005, n=7) and prolonged latencies by 22.3±4.3% (±S.D., P<0.0005, n=7). Application of serotonin (100 μM) decreased amplitudes by 6.6±5.0% (±S.D., P<0.05, n=6). Extracellular calcium concentration ([Ca 2+] e) was measured at various depths in the dorsal column with ion-selective microelectrodes. Four hours' superfusion with Ca 2+-free Ringer solution reduced [Ca 2+] e to less than 0.1 mM in dorsal columns. In 1.5 mM Ca 2+ Ringer solution, quipazine increased the amplitudes by 38.3±5.8% ( P<0.0005, n=6). Likewise, serotonin increased the amplitudes by 13.8±4.9% ( P<0.005, n=6). In contrast however, 8-hydroxy-dipropylaminotetralin still reduced amplitudes by 35.0±6.4% ( P<0.0005, n=7) and prolonged latencies by 24.1±4.5% ( P<0.0005, n=7). In experiment 2, we investigated calcium-dependent and cAMP-mediated protein kinase signalling pathways to evaluate their role as intracellular messengers for serotonin 2A receptor activation. Two protein kinase inhibitors, 50 μM H7 (an inhibitor of protein kinase C and c-AMP dependent protein kinase) and 100 μM d-sphingosine (an inhibitor of protein kinase A and C) effectively eliminated the excitatory effects of the serotonin 2A agonist. 100 μM cadmium (a Ca 2+ channel blocker) also blocked the effects of quipazine. Neither these protein kinase inhibitors nor cadmium alone affected action potential amplitudes. These results suggest that replacing Ca 2+ with Mg 2+ blocks the excitatory effects of quipazine but does not prevent the inhibitory effects of 8-hydroxy-dipropylaminotetralin, and calcium-mediated protein kinase mechanisms modulate axonal excitability changes induced by serotonin and its agonist.