Celecoxib is a selective cyclooxygenase-2-(COX-2)-inhibitor used to treat inflammation and pain and prevents colorectal cancer in patients at high doses by affecting several non-COX-2 proteins. However, celecoxib concentrations appropriate to inhibit proliferation or to induce apoptosis in cell culture (up to 100 microM) clearly exceed those in human plasma (up to 10 microM). Therefore, we speculated that celecoxib might accumulate in human cells, which may facilitate the drug's interaction with non-COX-2 proteins. Determination of intracellular celecoxib concentrations by liquid chromatography tandem mass spectrometry gave five- to tenfold higher levels as compared to other coxibs (etoricoxib, valdecoxib, lumiracoxib, and rofecoxib) in different tumor cell types, including human HCA-7 and HCT-116 colon carcinoma cells, BL-41 B lymphocytes, Mono Mac 6 monocytes, and in mouse NIH-3T3 non-tumor fibroblasts. This intracellular accumulation of celecoxib was due to an integration of the drug into cellular phospholipid membranes as demonstrated by nuclear Overhauser spectroscopy/nuclear magnetic resonance. Consequently, celecoxib disturbed the plasma membrane integrity of HCT-116 cells and displayed an increased COX-2-inhibitory potency in HCA-7 cells. The use of other coxibs demonstrated that intracellular accumulation is peculiar of celecoxib. Accumulation of celecoxib in human cells may provide a novel molecular basis for the ability of the drug to interact with non-COX-2 targets in vivo despite comparatively low plasma concentrations.