HL60 cells are human promyeloid cells that can be induced to differentiate by physiological stimuli (e.g. all-trans retinoic acid (ATRA), 1 α,25-dihydroxyvitamin D₃ (D₃), granulocyte colony-stimulating factor (G-CSF)) and by non-physiological agents such as dimethysulphoxide (DMSO) and protein kinase C-activating phorbol esters. The sensitivity of HL60 cells to physiological differentiating agents, but not to DMSO, is enhanced when cells are exposed to ‘anti-inflammatory agents’ (e.g. indomethacin) or are ‘primed’ (pretreated) with a small amount of ATRA: alone, neither treatment induces differentiation. We earlier suggested that indomethacin might act by inhibiting the endogenous formation of a differentiation-suppressing prostanoid (Bunce, C.M., et al. (1994) Leukemia 8, 595–604). Studies of the formation of prostanoids by HL60 cells and of the effects of prostanoids on these cells failed to identify any prostanoid that could be implicated in sensitization by indomethacin. 3α-Hydroxysteroid dehydrogenase (3α-HSD) is another target of such ‘anti-inflammatory agents’. Steroid inhibitors of 3α-HSD sensitized HL60 cells to inducers of differentiation in a manner similar to indomethacin. 3α-HSD is a member of the aldoketoreductase enzyme family, which comprises many enzymes of similar size and primary sequence. A protein that was recognised by an antiserum to 3α-HSD was found in HL60 cells, but the cells showed no detectable 3α-HSD activity. The 3α-HSD-like protein was strikingly down-regulated by ‘priming’ doses of ATRA. When treatment with a differentiation-sensitizing ‘anti-inflammatory agent’ or steroid was combined with ATRA ‘priming’, the effects of the different treatments were not additive: the resulting increase in sensitivity equalled that achievable by either treatment alone. We conclude that interference with a single intracellular regulatory mechanism underlies the increases in sensitivity of cells to differentiating agents that are caused by anti-inflammatory agents, by certain steroids and by ‘priming’ with ATRA. Decreased activity of a yet-to-be-identified member of the aldoketoreductase family of dehydrogenases is likely to be a central feature of a previously unrecognised mechanism that controls the responsiveness of cells to environmental stimuli such as retinoids and D₃.