The intracellular free Ca2+ concentration and membrane potential changes evoked by acetylcholine were recorded from whole-cell patch-clamped endothelial cells in situ in the isolated rat aorta. The endothelium had a resting membrane potential of -52 +/- 3 mV (SEM, range -35 mV to -76 mV n = 34) and a low input resistance (32 - 54 M omega). The membrane potential hyperpolarised by 3-30 mV on continuous application of acetylcholine at concentrations that produced endothelium-dependent relaxations in isolated rat aortic rings (range 1-500 nM). The response often comprised complex fluctuations of hyperpolarised membrane potential. Calcium concentration was measured with the fluorescent indicator furaptra, which has a wide range and minimises Ca2+ buffering. Acetylcholine evoked an initial rapid elevation of intracellular Ca2+ concentration, peaking in the range 6-35 microM, which declined with a half time of approximately 6 s, followed by repetitive [Ca2+] spikes of amplitude 2-18 microM in 23 of 34 cells. The initial [Ca2+] transient and hyperpolarisation were unaffected by removal of external Ca2+, whilst subsequent [Ca2+] spikes and maintained hyperpolarisations required the presence of external Ca2+. Both the hyperpolarisation and Ca2+ responses elicited by acetylcholine were abolished by atropine (1 microM). These results show that endothelial cells in situ exhibit large, fast repetitive [Ca2+] spikes in response to extracellular acetylcholine.