Celiac disease is associated with endomysial antibodies (EmA), which have recently been reported to be directed to tissue transglutaminase (tTG). To demonstrate binding of antibodies to recombinant tTG, human tTG was cloned, expressed by in vitro transcription/translation and used to develop novel radioligand assays for combined and single detection of immunoglobulin A (IgA) and G (IgG)-specific antibodies. IgA and IgG-tTGA were found in 43 (95.6%) of 45 patients with newly-diagnosed celiac disease verified by biopsy. In addition, all 30 sera from patients with gastrointestinal symptoms and positive EmA were positive for IgA-tTGA, and all but one serum (96.7%) had antibodies of the IgG class. Receiver-operating characteristic analysis including 574 sera from healthy controls revealed a specificity of 99.5%. By means of these new assays, we identified all patients with endomysial antibodies and achieved, at equal specificity, an even improved sensitivity (95.6%) as compared to EmA (91.1%) detected by the standard immunofluorescence test. Here, we have provided direct evidence that recombinant tTG is a major target of antibodies in celiac disease. Our data suggest that tTGA measured by radioligand assay have the power to overcome the limitations of the EmA-test. This new strategy may considerably facilitate large-scale screening for silent and latent celiac disease.