Using patch- and carbon-fiber electrodes, release phenomena of adenine nucleotides and serotonin from megakaryocytes isolated from the bone marrow of the mouse were studied. Megakaryocytes express ionotropic purinergic receptors on their surfaces. Under the condition of whole-cell recording, the cells showed spike-like spontaneous inward currents. The spontaneous currents were carried by cations and had amplitudes of 30-800 pA at -43 mV and durations of 0.1-0.3 s. Pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS; 100 µM) and suramin (100 µM), purinoceptor-blocking agents, depressed the currents reversibly. It is thought that the receptor involved was the P2X₁ subtype on the cell and that the currents were due to activation of the P2X₁ receptor by adenine nucleotides released from the cell. The currents showed a skewed amplitude distribution, suggesting variation of vesicular contents and/or distinct localization or varied density of receptors on the cell. Frequency of the spontaneous inward currents was enhanced by external application of platelet-activating substances, thrombin (0.4 U/ml), phorbol-ester (100 nM) and ADP (2 µM), at low concentrations. With a carbon-fiber electrode, which can detect oxidizable substances including serotonin, spike-like oxidation currents from the external surface of the megakaryocyte were detected. The frequency of the oxidation currents increased remarkably after the application of thrombin (10 U/ml). The majority of the oxidation currents coincided with the rising phase of the whole-cell currents, suggesting co-release of serotonin and adenine nucleotide from the same vesicle. It is concluded that megakaryocytes store adenine nucleotides and serotonin in the same vesicle and release them simultaneously in a discrete manner.