Neurons in organotypic hippocampal slice cultures (OHSCs) are known to preserve morphological and physiological features of the in vivo situation; however, little is known about the properties of microglial cells under these in vitro conditions. In this study, we addressed the question whether microglial cells in OHSCs are initially activated following explantation but return to a resting state during in vitro cultivation. Thus, we analyzed a) microglial cell morphology, b) microglial cell distribution, and c) expression of integrin adhesion molecules as putative markers of microglial activation. Hippocampal slices fixed immediately following explantation showed only resting microglial cells, mainly located in the paraventricular regions. After 3 days in vitro (div) OHSC surfaces were covered by activated microglia, whereas intermediate layers contained fewer microglial cells, giving the slices a sandwich-like appearance with the intact hippocampal formation being surrounded by glial tissue. After 3 div, microglial cells in intermediate layers of OHSCs showed activated morphology with ovaloid cytoplasm and no or merely few cytoplasmic processes; after 6 div, however, an increasing degree of ramification could be observed. After 9 div, microglia in intermediate layers had almost regained the morphological appearance of resting cells with filigrane cytoplasmic processes extended in all directions. The integrin adhesion molecules LFA-1 (alpha and beta chains) and VLA-4 were expressed on most microglial cells with activated morphology, as verified by co-localization with double immunofluorescence labeling for LFA-1 or VLA-4 and Griffonia simplicifolia isolectin B4 (GFS-B4). In contrast, only low levels of integrin adhesion molecule expression were also found on reactive astrocytes along slice surfaces. However, LFA-1 or VLA-4 were never found on ramified microglial cells, and double immunofluorescence labeling of LFA-1 or VLA-4 with ramified GFS-B4+ microglia never occurred. We conclude that a) originally resting microglial cells activated in an early phase of in vitro culture but regain a resting status after at least 6 div; and b) integrin adhesion molecules LFA1 and VLA-4 are potential markers of microglial activation, as they were found on activated but never on resting microglial cells. This enables further investigations on immunological and electrophysiological features of resting and activated microglial cells under in vitro conditions.