The mpkCCD_c14 cell line is derived from mouse cortical collecting duct, and expresses aquaporin 2 (AQP2) endogenously, in a vasopressin (VP)-regulated fashion (Hasler et al. 2002). In the present study we have investigated whether these cells shuttle AQP2 to the plasma membrane in response to VP. We have also investigated the effect of angiotensin II (AII), which we have previously shown to cause shuttling in collecting ducts. Cells were grown on Falcon cell culture inserts in modified DMEM:F-12 medium (Hasler et al. 2002), until transepithelial resistance was maximal. They were then kept for 24 h in serum- and hormone-free medium, followed by 24 h in the same medium supplemented with 1 nM VP. After a 1 h wash to cause internalisation of AQP2, cells were treated with nothing, VP, or AII (100 pM) for 30 min. For confocal microscopy, cells were then fixed, incubated with fluorescein-tagged wheat germ agglutinin to mark the apical surface, permeabilized, and labelled with an antibody against the c-terminus of AQP2, with a Cy3-tagged secondary antibody. For semi-quantitative measurements of AQP2 localisation, cells were homogenised for membrane fractionation and used for Western blotting. For measurement of cAMP levels, cells were lysed in 0.1 M HCl for use with the Sigma cAMP ELISA. Results were analysed using Student’s t tests and the false-discovery rate procedure, and data expressed as mean ± S.E.M. Western blotting demonstrated that AQP2 shuttles from intracellular vesicles to the plasma membrane in response to vasopressin (268 ± 44% vs. control, n=4, p<0.05). Confocal microscopy demonstrated that AQP2 was predominantly intracellular in control cells, and predominantly apical after VP. VP also caused an increase in cAMP levels (35 ± 5 pmol/ml vs. 11 ± 1 pmol/ml, n=6, p<0.05). Results indicate that treatment with AII did not cause a significant increase in the shuttling of AQP2, although it did increase slightly compared to controls. Confocal microscopy also showed some areas of apical AQP2 labelling with AII treatment, although some areas remained intracellular. However, AII did not cause an increase in cAMP levels. These results demonstrate that this cell line is a valuable tool for investigating the acute regulation of AQP2 distribution, as well as the long-term changes in expression previously described, and that AII may induce AQP2 shuttling to a very slight extent, although apparently not via cAMP formation.