It has been demonstrated previously that cannabinol (CBN) differentially modulates interleukin-2 (IL-2) protein secretion by T cells with a corresponding change in extracellular signal-regulated kinase activity. The objective of the present studies was to further investigate the molecular mechanism by which CBN enhances IL-2 gene expression using the EL4 T cell line. We demonstrate here that steady-state IL-2 mRNA expression was significantly enhanced by CBN in a concentration-dependent manner in EL4 cells activated with suboptimal concentrations of phorbol-12-myristate-13-acetate (2-10 nM). Concordantly, a marked increase was observed in nuclear factor of activated T cells (NF-AT) DNA binding activity to the IL-2 distal NF-AT site, but not to nuclear factor for immunoglobulin kappa chain in B cells or activator protein 1 motifs. Transient transfection of EL4 cells with a reporter gene under the control of multiple IL-2 distal NF-AT motifs exhibited increased transcriptional activity by CBN in suboptimally activated cells. In addition, the CBN-mediated enhancement of IL-2 protein secretion and the transcriptional activity of the IL-2 distal NF-AT reporter gene was abrogated by the calcium/calmodulin-dependent protein kinase inhibitor KN93, but not by the CB2 receptor antagonist SR144528. Enhancement of IL-2 was also demonstrated with CP55940, Delta(9)-tetrahydrocannabinol, and cannabidiol, thus suggesting that the phenomenon is not unique to CBN. Collectively, these results suggest that increased IL-2 secretion by CBN is mediated through the enhancement of IL-2 gene transcription by activation of NF-AT in a CB1/CB2-independent manner.