Glucocorticoid resistance is a serious clinical problem in chronic inflammatory diseases, because many patients with rheumatoid arthritis, asthma, or Crohn's disease fail to respond to steroid treatment. The molecular mechanisms underlying this unresponsiveness, however, are completely unknown. The effects of steroids are largely mediated by the interference of the glucocorticoid receptor (GR) with proinflammatory transcription factors. In the present study, we therefore investigated the activation of the transcription factors nuclear factor-kappaB (NF-kappaB), activator protein-1 (AP-1), and the upstream kinases p38 and c-Jun N-terminal kinase (JNK) in steroid-sensitive and steroid-resistant patients with Crohn's disease. We demonstrated that steroid-sensitive and steroid-resistant patients reveal a remarkably different cellular activation pattern of these proinflammatory mediators. In steroid-sensitive patients, activation of NF-kappaB, AP-1, p38, and JNK was mainly found in lamina propria macrophages. In contrast, steroid-resistant patients revealed activation of all these mediators mostly in epithelial cells. The functional interference of the proinflammatory mediators with the glucocorticoid response was supported by reporter gene assays. Expression of NF-kappaB and, interestingly, also JNK1 and p38 inhibited the activity of the GR. Thus, our results suggest that steroid resistance is associated with increased epithelial activation of stress-activated protein kinases and NF-kappaB, which might inhibit the anti-inflammatory action of a limited number of GRs.