Singlet oxygen (¹O₂) is believed to be the major cytotoxic agent involved in photodynamic therapy (PDT). Measurement of ¹O₂ near-infrared (NIR) luminescence at 1270 nm in biological environments is confounded by the strongly reduced ¹O₂ lifetime and probably has never been achieved. We present evidence that this is now possible, using a new NIR-sensitive photomultiplier tube. Time-resolved ¹O₂ luminescence measurements were made in various solutions of aluminum tetrasulphonated phthalocyanine (AlS₄Pc) and Photofrin. Measurements were also performed on suspensions of leukemia cells incubated with AlS₄Pc, and a true intracellular component of the ¹O₂ signal was clearly identified. Time-resolved analysis showed a strongly reduced ¹O₂ lifetime and an increased photosensitizer triplet-state lifetime in the intracellular component. In vivo measurements were performed on normal skin and liver of Wistar rats sensitized with 50 mg/kg AlS₄Pc. In each case, a small but statistically significant spectral peak was observed at 1270 nm. The ¹O₂ lifetime based on photon count rate measurements at 1270 nm was 0.03–0.18 μs, consistent with published upper limits. We believe that these are the first direct observations of PDT-generated intracellular and in vivo ¹O₂. The detector technology provides a new tool for PDT research and possibly clinical use.