A double immunofluorescence staining technique is described for differentiation between cell-attached (extracellular) and ingested (intracellular) bacteria by HEp-2 cells in cell culture monolayers. This method is based upon the observation that membranes of viable mammalian cells are impermeable for antibodies but are rendered permeable by treatment with fixatives. Consequently, extracellular bacteria can be stained by specific rhodamine-labeled antibodies before fixation, and intracellular bacteria can be visualized by treatment with specific fluorescein-labeled antibodies after fixation. The accuracy and simplicity of this method is demonstrated with HEp-2 cell culture monolayers as target cells and an isogenic pair of Yersinia enterocolitica, one of which is phagocytosis resistant and the other of which is phagocytosis sensitive. Furthermore, it is shown that this staining technique is also applicable for studying the interaction of bacteria with macrophages and fibroblasts.