Mycoplasma synoviae (MS) is an important pathogen of domestic poultry and is prevalent in commercial layers. During the last decade Escherichia coli peritonitis became a major cause of layer mortality. The possible role of MS in the E. coli peritonitis syndrome of laying hens was studied. Four groups of 64 mycoplasma-free commercial layers at the onset of lay (about 80% daily production) were challenged with a virulent MS strain or a virulent avian E. coli strain or both. The four experimental groups were identified as follows: negative control, E. coli, MS, and MS plus E. coli. A typical E. coli peritonitis mortality was reproduced and included one, three, zero, and five birds in the negative control, E. coli, MS, and MS plus E. coli groups, respectively. Only the increased mortality in the MS plus E. coli group had statistical significance. Four weeks postchallenge 10 clinically normal birds from each of the four experimental groups were necropsied. All of the examined birds in the two MS-challenged groups demonstrated severe tracheal lesions. Body cavity lesions were detected in two and four birds in the MS and MS plus E. coli groups, respectively. The results demonstrate a possible pathogenesis mechanism of respiratory origin with regard to the layer E. coli peritonitis syndrome, show the MS pathological effect in layers, and indicate that a virulent MS strain can act as a complicating factor in the layer E. coli peritonitis syndrome.