Relaxation of airway smooth muscle is dependent predominantly upon elevation of cell cyclic AMP content. Whilst the processes involved in the elevation of cell cyclic AMP content are reasonably well established, the mechanisms governing subsequent control of cyclic AMP turnover are less clear. Breakdown of cyclic AMP is solely regulated by phosphodiesterase isoenzymes. We have previously reported that phosphodiesterase 4 family members are likely to be important in this process, and that expression of phosphodiesterase 4D variants is actively regulated at the transcriptional level. Here we demonstrate a key role for phosphodiesterase 4D5 in the control of ₂ adrenoceptor-stimulated cyclic AMP activity in human airway smooth muscle cells using splice variant specific siRNA knockdown. Furthermore, we show, using an Epac-based cyclic AMP sensitive fluorescent probe that these intracellular cyclic AMP gradients are controlled both temporally and dynamically by phosphodiesterase 4D5. Elevation of cyclic AMP within the cytoplasm following ₂ adrenoceptor stimulation is rapid and shows no distinct spatial compartmentalization in these cells. These data suggest that phosphodiesterase 4D5, despite being a minor component of the tissue phosphodiesterase pool, is the key physiological regulator of ₂ adrenoceptor-induced cyclic AMP turnover within human airway smooth muscle.