Human fibroblast monolayers were applied as a nuclear substrate in indirect immunofluorescence for the detection of anti-nuclear antibodies (ANA). Comparison of the results obtained with this substrate and with conventional rat liver sections led to the conclusion that the application of human fibroblast monolayers as a substrate has the following advantages: 1) better recognition of the distinct nuclear staining patterns; 2) more convenient serum titrations, since a cryostat is not needed and twelve tests can be performed on one slide; 3) detection of significantly higher ANA litres in sera from patients with SLE and MCTD; 4) recognition of a type of ANA that was not detected on rat liver sections. These latter antibodies produced a discrete speckled pattern of fluorescence, which was completely lost after acid elution of the fibroblasts. Using HEp-2 cells in metaphase, it was shown that the antibodies were directed against the centromere regions of the chromosomes. These anti-centromere antibodies were detected in sera from patients with severe Raynaud's phenomenon. Underlying disorders were scleroderma (8 patients, 5 of them with CREST syndrome), Sjogren's syndrome (2 patients), and Raynaud's phenomenon in combination with a few symptoms of connective tissue diseases without fulfilling the criteria for a specific disease (5 patients).