In vivo, keratinocyte skin cells are exposed to photooxidative processes, some of which can be mediated by singlet molecular oxygen (1O2), a species that is very difficult to detect spectrally in cells. We photosensitized 1O2 in cultured HaCaT keratinocytes stained with rose bengal (RB) that localizes exclusively inside the keratinocyte hydrophobic regions, as evidenced by strongly red-shifted absorbance and intense fluorescence. We used keratinocytes grown in a monolayer on a plastic coverslip and in suspension. The phosphorescence spectrum (1200-1350 nm) from 1O2 was strongest when the coverslip containing RB-stained keratinocytes was irradiated in air. The spectral intensity decreased when the coverslip was immersed in D2O during irradiation and was almost completely quenched when it was irradiated while immersed in water. Water not only shortens the 1O2 lifetime but also reabsorbs part of the 1O2 phosphorescence, processes that do not occur when 1O2 is produced in a keratinocyte layer exposed to air. Because the RB was inside keratinocytes, singlet oxygen must also be produced inside the keratinocytes. However, the sensitivity to the extracellular environment suggests that most of the detectable 1O2 phosphorescence originates from those 1O2 molecules that escaped from the cell through its membrane into D2O or into the air, where 1O2 has longer lifetimes. Our results confirm directly that 1O2 is indeed photosensitized in living cells by RB. They also suggest that keratinocyte monolayers may be a good cell model to examine in vitro the production of 1O2 by other photosensitizers of environmental and photomedical interest.