In vivo anti-nuclear antibody (ANA) was observed by direct immunofluorescence microscopy in epithelial cell nuclei in forty-four biopsies from thirty-three patients. The tissue containing the ANA was macroscopically normal in twenty-seven patients. The thirty-three patients with in vivo biopsy ANA included twenty-three with SLE, three with mixed connective tissue disease, two each with multi-system Sjögren's syndrome, dermatomyositis, and progressive systemic sclerosis, and one with rheumatoid arthritis. Features of sicca syndrome were noted in seventeen patients. The patterns of the in vivo biopsy ANA in the thirty-three patients were speckled (21), homogeneous (6), nodular (2), and both speckled and homogeneous (4). Complement was not detected in the epithelial cell nuclei. Immunoglobulin(s) and/or complement were deposited along the dermoepidermal junction in thirty-two of the forty-four biopsies, and in dermal blood vessels in twenty-two biopsies. Each patient had serum ANA against rat liver substrate; twenty-seven had high titre ANA (1 in 1000 or greater). Elevated levels of DNA-binding were found in twenty patients (61%), but the level of DNA-binding did not correlate with the intensity of in vitro biopsy ANA staining. Serum antibody to ribonucleoprotein (RNP) was present in eight of the twenty-three patients tested (35%), all eight patients having clinical features of sicca syndrome. Hypocomplementaemia was found in thirteen patients (40%), all of whom had active SLE. In vivo biopsy ANA appears to be a real phenomenon of unknown aetiology, and not an artifact, which is found in some patients with active multisystem autoimmune disease, especially SLE.