The association between ATP depletion and enzyme release from cells has been described in two different ways: as a more or less linear dependence, or with a threshold value below which the enzyme release will start. We have investigated the association between ATP depletion caused by various metabolic inhibitors and enzyme release on quiescent fibroblasts. We found that the enzyme release never started before the ATP had decreased to a critical low level. Addition of glucose to cells while ATP was still above this critical level led to a regeneration of ATP and enzyme release did not occur. If ATP was lowered to 35-40% and kept there for 24 h, the enzyme release was minimal. These results support the threshold theory for release of enzymes from cells.