Cyclic adenosine 3′,5′-monophosphate (cAMP) was originally shown to induce gene transcription through activation of cAMP-dependent protein kinase (PKA), and subsequent phosphorylation of the transcription factor cAMP response element-binding protein, CREB, at serine-133. However, elevated cAMP levels may activate multiple signalling pathways with protein kinases that can phosphorylate CREB at serine-133. We analysed the pathways involved in CREB phosphorylation and activation in NIH 3T3 cells exposed to the cAMP elevating agent forskolin. PKA represented the predominant pathway during the burst phase, while the mitogen-activated protein kinase p38 pathway became activated in a PKA-dependent fashion in forskolin treated cells. The phosphorylation kinetics of p38 was delayed compared to PKA activation. Activated p38 stimulated CREB-mediated transcription and potentiated the transcriptional strength of CREB provoked by forskolin. The p38-mediated activation of CREB was inhibited by dominant negative mutants of MSK-1 and by the PKA/MSK-1 inhibitor H89, but not by dominant negative mutants of MSK-2/RSK-B and MAPKAPK2. Our results suggest that forskolin-induced CREB phosphorylation and activation in NIH 3T3 cells is mediated directly by PKA and by a time-delayed PKA-dependent p38/MSK-1 pathway. This bifurcation and time-dependent regulation of the cAMP-responsive signalling pathways may enable the cell to endure and/or enforce a cellular response provoked by a cAMP-elevating stimulus.