Expression of cytokines by T lymphocytes is a highly balanced process, involving stimulatory and inhibitory intracellular signalling pathways. In the present work, we attempted to clarify the role of cAMP on interferon-gamma (IFN-gamma), interleukin (IL)-10, IL-4 and IL-13 expression as well as on the inducible nitric oxide synthase (iNOS) expression. Treatment of phytohaemagglutinin (PHA)/phorbol 12-myristate 13-acetate (PMA)-activated Jurkat cells with either dibutyryl-cyclic adenosine monophosphate (cAMP) or pentoxifylline induced a strong inhibition of IFN-gamma mRNA expression as measured by reverse transcription (RT)-polymerase chain reaction (PCR), without affecting IL-10 expression. Both cholera toxin and prostaglandin E2 (PGE2) induced a strong inhibition of IFN-gamma mRNA expression, whereas IL-10 mRNA expression was significantly enhanced. This differential regulation of IFN-gamma and IL-10 expression was related to intracellular cAMP concentration. IL-13 and IL-4 mRNA expressions were not inhibited. We developed a new method based on immunofluorescence for intracellular cytokine detection followed by optical and computerized image processing, and our results showed that IFN-gamma protein was strongly inhibited when cells were treated with PGE2 or dibutyryl (db)-cAMP, whereas IL-10 protein was enhanced. This suggests that cAMP exerts its action at both the transcriptional and protein levels. iNOS mRNA expression was markedly elevated in the presence of PGE2. The generation of nitric oxide using sodium nitroprusside (SNP) induced a dramatic decrease of IFN-gamma, while IL-10 was enhanced; and conversely the inhibition of iNOS activity using 1-NG-monomethyl arginine (1-NMMA) induced a clear inhibition of IL-10 and IL-4, while IFN-gamma was enhanced. These results provide evidence that the protein kinase A (PKA) activation pathway plays a prominent role in the balance between the type 1 and type 2 cytokine profile in PHA/PMA-activated Jurkat cells. Data also suggest that iNOS expression is under the control of PKA activation, and that NO seems to be able to assume the polarization of activated T cells to the type 2 profile.