Translocation from the outer to the inner membrane leaflet (flip) of phospholipids after ultraviolet A (UVA) irradiation was investigated in Chinese hamster ovary cells. Fluorescent 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphoserine (NBD-labeled phosphatidylserine [NBD-PS]) was used to assay transbilayer lipid movement. A marked increase in flip of NBD-PS was observed immediately after low-dose UVA irradiation which was not lethal and returned to the basal level after 6 h. UVA-induced flip was not attributed to the increase of permeability by UVA irradiation because cells that were negative for staining with propidium iodide also showed increased flip of NBD-PS. Furthermore, the enhancement was independent of adenosine 5′-triphosphate, demonstrating the lack of involvement of phospholipid translocase. Marked increases were also observed in flip of both NBD-phosphatidylethanolamine and NBD-phosphatidylcholine immediately after UVA irradiation, showing that the increase was independent on the head groups of phospholipids. These findings indicated that UVA changes the flip–flop of phospholipids and that the cell membrane is a molecular and cellular target of UVA.