The cAMP analogue dibutyryl cAMP (dbcAMP) is often used to activate the protein kinase A pathway and to study the expression of cAMP-responsive genes. Here we show that in NIH 3T3 cells dbcAMP is able to activate PKA, but fails to stimulate expression of the cAMP-inducible c-fos gene. Co-expression of A-kinase anchoring protein 75, previously shown to amplify cAMP signalling and to stimulate c-fos expression, could not restore cAMP responsiveness of the c-fos promoter. DbcAMP-induced activation of PKA may result in poor translocation of the catalytic sub-units of PKA to the nucleus, indicated by the lack of both Ser-133 phosphorylation of the cAMP-response element binding factor CREB and stimulation of the transcriptional activity of this factor. DbcAMP treatment, however, inhibited cell proliferation. These results suggest that cAMP-mediated inhibition of proliferation may be independent of translocation of the catalytic sub-units into the nucleus.