Exocytosis and intracellular free calcium ([Ca2+]in) were simultaneously recorded in single human neutrophils using patch-clamp capacitance measurements and the fura-2 fluorescence ratio method. Intracellular application of guanosine-5′-O(3-thiotriphosphate) (GTPγS) stimulates both exocytosis and a calcium transient. The calcium transient starts to develop after a lag phase of ∼40s and normally appears to trigger the onset of exocytosis indicated by the beginning of the capacitance increase. After this delay [Ca2+]in increases from ∼150 nM to ∼600 nM with a sigmoidal time course. The peak concentration is reached within ∼30 s but the main increase occurs during ∼ 3s. [Ca2+]in subsequently decays within 1–2 min to a level which is close to the resting value. This calcium transient is due to calcium release from inositoltrisphosphate-sensitive intracellular stores. Exocytosis also occurs if the calcium transient is abolished by intracellular EGTA but the lag phase is markedly prolonged. The GTPγS-induced calcium transient is very similar to that observed after stimulation with N-formyl-methionyl-leucyl-phenylalanine. The interplay between guanine nucleotides, [Ca2+]in and exocytosis in neutrophils closely resembles previous results obtained in mast cells suggesting a similar regulation of exocytosis in both cell types.