Early during the process of apoptosis, cells lose their phospholipid membrane asymmetry and expose phosphatidylserine (PS) at the cell surface while maintaining their plasma membrane integrity intact. This process can be monitored for suspended cell types by using annexin V-FITC, which is a Ca(2+)-dependent, phospholipid-binding protein with high affinity for PS, and flow cytometry. If adherent cell types are to be studied for this apoptosis-associated phenomenon, then a problem is encountered, in that specific membrane damage occurs during harvesting. In this paper, a flow cytometric-based method is described that allows the measurement of loss of phospholipid asymmetry during apoptosis of adherent cells in culture. The method relies on the phospholipid binding property of biotinylated annexin V. Furthermore, the use of this conjugate allows tricolor flow cytometric analysis of apoptosis. Employing the method to MR65 cells, which were initiated by olomoucine to enter apoptosis, it is shown that PS exposure occurs early after the onset of apoptosis and, at the prevalent time-resolution, that PS exposure is accompanied by loss of both cytokeratin and DNA. The annexin V+ cells appear as a characteristic sub-G1 peak in the DNA histogram.