Microtubules (MT) are dynamic, polar structures which mediate a number of essential cellular functions, including vesicle transport. In Madin-Darby Canine Kidney cells, MT networks reorganise as the cells become polarized (Bacallao et al, 1989), with their minus ends in the apical pole. Consistent with this, vesicles carrying aquaporin 2 (AQP2) to the apical surface of collecting duct cells have dynein, a minus-end directed motor, attached. We investigated MT organisation in mpkCCDc14 cells, a cell line derived from murine cortical collecting duct principal cells (Bens et al 1999). Cells were seeded onto both coverslips and polycarbonate filters, and grown in modified DMEM medium. In some experiments cells were treated with 1nM desmopressin (dDAVP) for 4-5 days to induce AQP2 expression. After three hours washout, half the cells were treated with dDAVP for 30 minutes to induce shuttling. Cells were fixed with either ice cold methanol (-20 °C) or 4% paraformaldehyde, fluorescently labelled using antibodies against α tubulin, γ tubulin, acetylated tubulin, AQP2 and end binding protein 1 (EB1) in various combinations, and viewed using deconvolution and confocal microscopy. All findings described below were confirmed on at least 2 independent preparations. As expected, cells grown on coverslips were not polarised, did not express AQP2, and displayed a fibroblastic appearance. Labelling for α tubulin revealed a basket weave pattern with the MT organised around the nucleus. Polarisation was seen in the filter-grown cells, although a proportion of fibroblastic cells were also present. Polarised cells were considerably taller than the fibroblastic cells. Labelling for α tubulin revealed a meshwork of MT in the apical part of the cell; microtubules could also be seen running down the lateral borders of the cell alongside the nucleus. Gamma tubulin, found in the centrioles and microtubule organising centre (MTOC), was located apically, usually as two foci. Co-labelling for γ and acetylated α tubulin (characteristic of cilia) confirmed the presence of primary cilia, with an associated basal body. After acute dDAVP treatment, labelling with EB1, which associates with microtubule plus ends, was most abundant in an apical meshwork, but was also seen at the bottom of the cell. Cells not acutely treated with dDAVP had a similar pattern, but a less prominent apical network, suggesting fewer new MTs forming in this region. In fibroblastic cells plus ends were located throughout, with no clear focus. AQP2 labelling was predominantly apical in cells acutely treated with dDAVP. Our results are consistent with the hypothesis that microtubule assembly is initiated primarily in the apical part of the cells, with plus ends projecting towards the basolateral membrane. Hence dynein will move AQP2-containing vesicles towards the apical surface.