The reliable identification of astrocytes for physiological measurements was always time-consuming and difficult. Recently, the fluorescent dye sulforhodamine 101 (SR101) was reported to label cortical glial cells in vivo [Nimmerjahn A, Kirchhoff F, Kerr JN, Helmchen F. Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo. Nat Methods 2004;1:31-7]. We adapted this technique for use in acute rat hippocampal slices at early postnatal stages (P3, 7, 15) and in young adults (P24-27) and describe a procedure for double-labeling of SR101 and ion-selective dyes. Using whole-cell patch-clamp, imaging, and immunohistochemistry, we characterized the properties of SR101-positive versus SR101-negative cells in the stratum radiatum. Our data show that SR101, in contrast to Fura-2 or SBFI, only stains a subset of glial cells. Throughout development, SR101-positive and SR101-negative cells differ in their basic membrane properties. Furthermore, SR101-positive cells undergo a developmental switch from variably rectifying to passive between P3 and P15 and lack voltage-gated Na+ currents. At P15, the majority of SR101-positive cells is positive for GFAP. Thus, our data demonstrate that SR101 selectively labels a subpopulation of glial cells in early juvenile hippocampi that shows the typical developmental changes and characteristics of classical astrocytes. Owing to its reliability and uncomplicated handling, we expect that this technique will be helpful in future investigations studying astrocytes in the developing brain.