Expression of cytokines by T lymphocytes is a highly balanced process, involving stimulatory and inhibitory intracellular signaling pathways. We have examined the modulating effects of the cAMP-dependent signaling pathway on the expression of interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in activated human T lymphocytes. 2'-O-dibutyryl-cAMP (db-cAMP), prostaglandin E2 (PGE2), isoproterenol (ISO), and isobutyl-methyl-xantin (IBMX) costimulated with concanavalin A (Con A) or Con A plus the phorbolester phorbol myristate acetate (PMA) inhibited IL-3 and GM-CSF mRNA accumulation compared to the effects of Con A or Con A plus PMA alone. Nuclear run-on experiments revealed that the inhibitory effect of db-cAMP could partially be ascribed to a five-fold reduction in transcription rate of both the IL-3 and GM-CSF gene in the presence of Con A or Con A plus PMA. mRNA stability studies demonstrated that PMA increased the stability of both transcripts. db-cAMP did not affect the stability of IL-3 and GM-CSF mRNAs in Con A activated cells. In contrast, in Con A plus PMA activated cells, db-cAMP significantly reduced the half-life of both transcripts: IL-3 >240 minutes vs. 90 minutes and GM-CSF 90 minutes vs. 60 minutes. Finally, in accordance with the mRNA data, db-cAMP, PGE2, and ISO reduced the secretion of IL-3 and GM-CSF protein in Con A and Con A plus PMA activated cells. In conclusion, these data demonstrate that the protein kinase A (PKA)-dependent signaling pathway is an important regulatory mechanism in controlling IL-3 and GM-CSF gene expression in activated human T lymphocytes.