A mechanosensitive Ca(2+)-permeable cation channel was recorded by patch clamp in isolated rat aortic endothelial cells. A low level of channel activity could be observed after seal formation. The channel displayed some inward rectification and had a conductance for inward current of approx. 32 pS in Ca(2+)-free pipette and bath solutions. Negative suction of -10 to -20 mmHg increased the probability of the channel being open. When the negative pressure in the pipette was raised to -35 to -45 mmHg, the channel underwent an abrupt transition to a large conductance substate that was interrupted occasionally by two other low conductance levels. Under this condition, the overwhelming majority of openings and closings were between a main level of 83 pS and the closed level. Compared to the 32 pS substate, the 83 pS large conductance substate had shorter mean open and closed times. The two channel substates had similar ionic selectivity and both were sensitive to the inhibition of cGMP and protein kinase G. This is the first demonstration showing that mechanostress can change the single channel conductance level of an ion channel in eukaryotic cells.