Although many membrane components have been described to be involved in the activation of cells by bacterial lipopolysaccharide (LPS), the question remains whether LPS, once internalized by target cells, is also capable of interacting with cytoplasmic elements in such a way that activation of cells results independently of receptor engagement. This is an important aspect to consider with respect to the development of strategies aimed at attenuating adverse effects of LPS in the framework of bacterial infections. In this study, human monocyte derived macrophages as representatives of one of the primary target cells activated by LPS, were microinjected with LPS to circumvent exogenous LPS stimulation. Parameters correlating to cytoplasmic activation of the nuclear transcription factor NFkappaB (intracellular calcium mobilization), to nuclear translocation of the NFkappaB p65 subunit and to mRNA-transcription of inflammatory cytokines known to be expressed upon exogenous LPS-stimulation and to require NFkappaB activation (interleukin-1beta, interleukin-6, tumor necrosis factor alpha) were investigated. In addition, the LPS-reporter cell line 3E10, which contains a reporter gene under the control of an NFkappaB-inducible promoter was analyzed with respect to NFkappaB nuclear translocation and reporter gene expression. None of the cellular systems used and none of the parameters investigated led to the observation that intracellular LPS leads to activation of the cells in comparison to external LPS stimulation. These experiments allow the conclusion that LPS in the cytoplasmic compartment does not lead to NFkappaB translocation, cytokine mRNA transcription, and NFkappaB dependent protein expression and suggest that these activation parameters require the interaction of LPS with external membrane components.