Sulforhodamine 101 (SR101) is widely used as a marker of astrocytes in the cortex and hippocampus in vivo and more recently also in acute brain slices. In this study we tested if SR101 is also an effective marker for astrocytes in the respiratory network of the brainstem. Using 2-photon microscopy we compared astrocytic labeling by transgenic expression of EGFP (TgN(hGFAP-EGFP) mice) with the degree of astroglial SR101-labeling in acute slices from hippocampus and brainstem. While SR101 was efficiently labeling EGFP- expressing astrocytes in hippocampus slices, we found that SR101-staining in the brainstem was very weak and also not cell type specific. Time-lapse 2-photon imaging experiments were performed to compare the time course of staining and un-staining of SR101 in hippocampus and brainstem astrocytes. In the hippocampus, both astrocytes and neurons were showing SR101-loading 5 min after starting the SR101 superfusion. However, SR101 was removed from the neurons during washout, while it was retained in astrocytes. In the brainstem, however, only a transient SR101-labeling was observed. To elucidate the labeling mechanism, we also tested if application of MK-571 to block multi-drug resistance transporters, changes SR101 staining of brainstem astrocytes. However, SR101 labeling remained restricted to EGFP-negative cells. Our data reveal a regional heterogeneity of astrocytes in hippocampus and brainstem. Further, we found no evidence that MDR transporters are preventing effective SR101 labeling of astrocytes in the brainstem. We conclude that SR101 cannot be used for selective staining of astrocytes in the brainstem.