Since the original synthesis of fluorescein in 1871, the use of xanthene fluorophores has remained ubiquitous in biology, medicine, and chemical biology. The wide utility of fluorescein and related xanthene dyes is due, in part, to their high brightness and wide range of colors available with simple modifications to the terminal and bridgehead atoms of the xanthene fluorophore core. By comparison, substitution of the pendant carboxylate has remained relatively underexplored. The identity of the substitution at this 3- position can have profound effects on the properties of xanthene fluorophores. We envisioned installation of a biologically relevant phosphonate functionality may provide access to fluorophores with unique properties, such as enhanced water solubility and opportunities for functionalization. Herein we will discuss the first syntheses of novel phosphonated xanthene fluorophores, their associated spectroscopic properties, and the utility of these dyes for both intracellular imaging and membrane potential sensing in living cells.