Regulation of adenylyl cyclases (ACs) by Ca2+ requires capacitative Ca2+ entry (CCE) (Cooper, D. M. F. (2003) Biochem. J. 375, 517-529), but whether Ca2+-sensitive phosphodiesterases (PDEs) are similarly discriminating has never been addressed. In the present study, a variety of conditions were devised to manipulate [Ca2+]i so that we could ask whether PDE1 selectively responds to different modes of elevating [Ca2+]i, viz. Ca2+ released from intracellular stores and various modes of Ca2+ entry. In 1321N1 human astrocytoma cells, the endogenous PDE1 (identified as PDE1A by reverse transcriptase-PCR) was largely insensitive to Ca2+ released from carbachol-sensitive stores but was robustly stimulated by a similar rise in [Ca2+]i due to carbachol-induced Ca2+ influx. Gd3+, which effectively blocked thapsigargin-induced CCE and its effect on PDE1A, also inhibited the activation of PDE1A by carbachol-induced Ca2+ entry. However, non-selective ionomycin-mediated Ca2+ entry also activated PDE1A, so that, unlike Ca2+-sensitive ACs, PDE1A cannot discriminate between the different sources of Ca2+ entry. Fractionation of the cells revealed that the Ca2+-calmodulin-stimulated PDE activity was not present at the plasma membrane but was associated with the cytosol and the organellar compartments of the cell. Therefore, the apparent disparity between PDE1A and ACs is likely to be the consequence of their differential subcellular localization. Nevertheless, in a physiological context, where artificial modes of elevating [Ca2+]i are not available, as with ACs, a dependence on CCE would be evident, and it would be the duration of this influx of Ca2+ that would determine how long PDE1A was activated.