An association between celiac disease (CD) and other autoimmune diseases such as connective tissue diseases (CTD), inflammatory bowel diseases (IBD), and primary biliary cirrhosis (PBC) has been reported in several studies. However, a high rate of false positives in autoantibody testing was noted, especially when tissue transglutaminase (tTG) from guinea pig liver was used. Thus, the real prevalence of CD in CTD, IBD, and PBC is unclear. In a case-control study, 400 patients with CTD, 170 with IBD, 48 with PBC, and 120 healthy subjects were investigated for CD by the analysis of IgA and IgG tTG antibodies using the more specific human recombinant tTG immunoenzymatic assay. Patients and controls with positive findings were further tested for antiendomysial antibodies by indirect immunofluorescence and HLA typing, and those found positive by either of these tests underwent duodenal biopsy to confirm a possible diagnosis of CD. Twelve patients were positive for IgA or IgG tTG antibodies, showing an overall prevalence of 1.9%. Only 1 healthy subject (0.8%) had a low level positive reaction for IgA anti-tTG. Among the 12 patients and the healthy subject, only 2 (1 SLE and 1 ulcerative colitis patient) were subsequently confirmed to be affected with CD by positive EMA, HLA, and small bowel biopsy findings. The highest rate of false positives was found in PBC patients (10.4%). For these reasons, serological screening testing for CD is not recommended in CTD patients or in subjects affected with IBD or PBC, unless there is a relevant clinical suspicion of CD.