The effects of various neurotransmitters on phosphoinositide hydrolysis, mobilization of Ca<sup>2+</sup> and release of [<sup>3</sup>H]-norepinephrine ([<sup>3</sup>H]-NE) were studied in cultures of sympathetic neurons of chick embryos. [<sup>3</sup>H]-inositol-1,4,5-triphosphate ([<sup>3</sup>H]-IP<sub>3</sub>) was increased in sympathetic neurons by acetylcholine (ACh), muscarine and serotonin (5-HT). Dopamine and norepinephrine did not stimulate phosphoinositide hydrolysis. Intracellular concentration of free Ca<sup>2+</sup> ([Ca<sup>2+</sup>]<sub>i</sub>) was measured in Indo-1-loaded sympathetic neurons at rest and after addition of test agents. Measurements were made in the cell body and growth cone regions since Ca<sup>2+</sup> mobilization is known to be different in different regions of the sympathetic neurons. ACh (nicotinic component was blocked by hexamethonium) and 5-HT failed to increase the [Ca<sup>2+</sup>]<sub>i</sub>, in the cell body as well as in the growth cone. The spontaneous release of [<sup>3</sup>H]-NE was not affected by ACh and 5-HT. Caffeine increased the [Ca<sup>2+</sup>]<sub>i</sub> only in the cell body but not in the growth cone and had no effect on the release of [<sup>3</sup>H]-NE. These results suggest that an IP3-insensitive but caffeine-sensitive pool of Ca<sup>2+</sup> is present only in the somatic region of sympathetic neurons and is not coupled to the transmitter release.