Reactive oxygen species like hydrogen peroxide (H₂O₂) have been shown to serve as messengers in the induction of NF‐κB and then in the activation and replication of HIV‐1 in human cells. Because singlet oxygen (¹O₂) is another very important reactive oxygen species whose action in transcription factor activation is totally undetermined, we started to investigate its role in both NF‐κB and HIV‐1 activation. For provoking unbalanced redox conditions, ¹O₂ was generated by photosensitization using methylene blue as photosensitizer. Lymphocytes or monocytes (ACH‐2 or U1 respectively) latently infected with HIV‐1 were treated by photosensitization mediated by methylene blue and the production of reactive oxygen species was monitored through their cytotoxic effect in infected cells. The generation of ¹O₂ by methylene blue turns out to be very efficient in inducing NF‐κB as a heterodimer composed of the p50 and p65 subunits. This induction appears specific since other transcription factors like AP‐1 are only weakly activated by this treatment. In comparison with other inducing treatments such as phorbol esters or tumor necrosis factor alpha (TNF‐α), the methylene‐blue‐mediated activation of NF‐κB is slow, becoming optimal 180 min after treatment. These kinetic data were obtained by following, on the same samples, both the emergence of NF‐κB in the nucleus and the disappearance of IκB‐α in the cytoplasmic extracts. Conjugated with the induction of this transcription factor, HIV‐1 reactivation from these latently infected cells was also observed by the measurement of reverse transcriptase activity in the cell supernatants. These data allow us to postulate that ¹O₂ is a biologically important reactive oxygen species which could play a role in the establishment of oxidative stress conditions leading to HIV‐1 activation via the presence of NF‐κB in the nucleus of infected cells.