Stimulation of neuropeptide Y (NPY) Y2 receptors induced an intracellular free Ca2+ ([Ca2+]i) increase in a human neuroblastoma cell line, CHP-234. When NPY in a Ca(2+)-free solution was applied, this increase was abolished. Depolarization with high KCl evoked no response, suggesting that the responses were not mediated by voltage-gated Ca2+ channels. There was no evidence that the NPY response consisted of a capacitative Ca2+ entry sensitive to internal Ca2+ store levels. The [Ca2+]i elevation was diminished by Ni2+, a blocker of Ca2+ entry. Mn2+ induced a quench of the fura-2 fluorescence, which ceased promptly upon the removal of NPY, indicating that Ca2+ entry was linked tightly to receptor activation. Although thapsigargin- and ryanodine-sensitive Ca2+ stores were present, NPY-induced responses were not impaired by pretreatment with either drug. Furthermore, NPY had no effect on the thapsigargin-sensitive store. Pertussis toxin did not affect the NPY-stimulated [Ca2+]i increase, although it abolished the NPY-dependent inhibition of cAMP production. It is concluded that the Y2 receptors couple directly to receptor-operated Ca2+ channels without the involvement of intracellular Ca2+ stores. The results also indicate that Y2 receptors can activate both pertussis toxin-sensitive and -insensitive mechanisms in the same cell.