Isoeugenol is a naturally occurring methoxyphenol found in a variety of foods and essential oils. We investigated the effect of isoeugenol on T-cell function and the regulatory mechanism underlying its effect. Isoeugenol and its structural analog eugenol suppressed the lymphoproliferative response to concanavalin A stimulation in B6C3F1 mouse splenocyte cultures. Isoeugenol inhibited phorbol 12-myristate 13-acetate (PMA) plus ionomycin (Io)-induced IL-2 mRNA expression and protein secretion in B6C3F1 mouse splenocytes, and in EL4.IL-2 mouse T-cells, as determined by real-time RT-PCR and ELISA, respectively. To further characterize the inhibitory mechanism of isoeugenol at the transcriptional level, we examined the DNA binding activity of the transcription factors for IL-2 using an electrophoretic mobility shift assay. Isoeugenol decreased the binding activity of NF-AT and NF-kappaB in PMA/Io-stimulated EL4.IL-2 cells, but no significant effect was observed for AP-1 or Oct binding activity. Western blot analysis showed that isoeugenol also decreased the nuclear translocation of cytoplasmic NF-AT and NF-kappaB. These results suggest that isoeugenol suppresses IL-2 production through a decrease of IL-2 mRNA expression and that the inhibition is mediated, at least in part, through the down-regulation of NF-AT and NF-kappaB.