Autoantibodies give us useful informations for the understanding of the co-existence of multiple autoimmune diseases in an individual patient. This study was undertaken to clarify the frequency of anti-centromere antibodies in the sera which were positive for anti-mitochondrial antibodies. Serum samples with anti-mitochondrial antibodies were examined on the frozen rat liver section and HEp-2 cells by indirect immunofluorescence. 57 out of 239 anti-mitochondrial antibody positive sera were found to contain specific autoantibodies reactive with centromeric antigens of the HEp-2 chromosomal spreads. The co-existence of anticentromere antibodies with anti-mitochondrial antibodies were significantly more frequent than with other defined autoantibodies. Clinical observation which shows frequent association of CREST syndrome and primary biliary cirrhosis appear to be explained by the frequent occurrence of anti-centromere antibodies and anti-mitochondrial antibodies.