1. Transcription factors such as NF-kappa B provide powerful targets for drugs to use in the treatment of cancer. In this report parthenolide (PT), a sesquiterpene lactone of herbal remedies such as feverfew (Tanacetum parthenium) with NF-kappa B inhibitory activity, markedly increased the degree of human leukaemia HL-60 cell differentiation when simultaneously combined with 5 nM 1 alpha,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)). PT by itself did not induce HL-60 cell differentiation. 2. Cytofluorometric analysis indicated that PT stimulated 1,25-(OH)(2)D(3)-induced differentiation of HL-60 cells predominantly into monocytes. 3. Pretreatment of HL-60 cells with PT before the 1,25-(OH)(2)D(3) addition also potentiated the 1,25-(OH)(2)D(3)-induced HL-60 cell differentiation in both a dose- and a time-dependent manner, in which the enhanced levels of cell differentiation closely correlated with the inhibitory levels of NF-kappa B binding activity by PT. 4. In contrast, santonin, a sesquiterpene lactone without an inhibitory activity of NF-kappa B binding to the kappa B sites, did not enhance the 1,25-(OH)(2)D(3)-induced HL-60 cell differentiation. 5. In transfection experiments, PT enhanced 1,25-(OH)(2)D(3)-induced VDRE-dependent promoter activity. Furthermore, PT restored 1,25-(OH)(2)D(3)-induced VDRE-dependent promoter activity inhibited by TNF-alpha, an activator of NF-kappa B signalling pathway. 6. These results indicate that PT strongly potentiates the 1,25-(OH)(2)D(3)-induced HL-60 cell differentiation into monocytes via the inhibition of NF-kappa B activity and provide evidence that inhibition of NF-kappa B activation can be a pre-requisite to the efficient entry of promyelocytic leukaemia cells into a differentiation pathway.