Impedance measurements in whole lenses showed that lens fiber cells possess different permeability properties to the epithelial cells from which they differentiate. To confirm these observations at the cellular level we analyzed the membrane properties of fiber cells isolated in the presence of the non-selective cation channel inhibitor Gd³⁺. Isolated fiber cells were viable in physiological [Ca²⁺] and exhibited a range of lengths that reflected their stage of differentiation. Analysis of a large population of fiber cells revealed a sub-group of cells whose conductivity matched values measured in the whole lens (Baldo and Mathias 1992). In this group of cells membrane resistance (R_m), conductivity (&#961;s) and reversal potential (E_rev) all varied with cell length, suggesting that the process of differentiation is associated with a change in the membrane properties of fiber cells. Using pharmacology and ion substitution experiments we show that newly differentiated fiber cells (<150µm) contained variable combinations of Ba²⁺-and TEA-sensitive K⁺ currents. Longer fiber cells (150 to 650µm) were dominated by a lyotropic anion conductance, which also appears to plays a role in the intact lens . Longer cells also exhibited a low-level, non-selective conductance that was eliminated by the replacement of extracellular Na⁺ with N-methyl D-glucamine, indicating that the lens contains both Gd³⁺-sensitive and -insensitive non-selective cation conductances. Fiber cell differentiation is therefore associated with a shift in membrane permeability from a dominant K⁺ conductance(s) towards larger contributions from anion and non-selective cation conductances as fiber cells elongate.