Ammonium uptake into the cell is known to be mediated by ammonium transport (Amt) proteins, which are present in all domains of life. The physiological role of Amt proteins remains elusive; indeed, loss-of-function experiments suggested that Amt proteins do not play an essential role in bacteria, yeast, and plants. Here we show that the reverse holds true in the tunicate Ciona intestinalis. The genome of C. intestinalis contains two AMT genes, Ci-AMT1a and Ci-AMT1b, which we show derive from an ascidian-specific gene duplication. We analyzed Ci-AMT expression during embryo development. Notably, Ci-AMT1a is expressed in the larval brain in a small number of cells defining a previously unseen V-shaped territory; these cells connect the brain cavity to the external environment. We show that the knockdown of Ci-AMT1a impairs the formation of the brain cavity and consequently the function of the otolith, the gravity-sensing organ contained in it. We speculate that the normal mechanical functioning (flotation and free movement) of the otolith may require a close regulation of ammonium salt(s) concentration in the brain cavity, because ammonium is known to affect both fluid density and viscosity; the cells forming the V territory may act as a conduit in achieving such a regulation.