Distinct profiles of autoantibodies directed to intracellular antigens can be detected in the systemic connective tissue diseases. They aid in establishing the correct diagnosis and are included in many sets of diagnostic criteria, such as the ones developed for systemic lupus erythematosus (anti-Smith antigen and anti-double-strand DNA antibodies), mixed connective tissue disease (anti-U1-nuclear ribonucleoprotein antibodies), and Sjögren's syndrome (SS) (anti-SS-A/Ro and anti-SS-B/La antibodies). They are useful prognostic markers in some situations and facilitate clinical and treatment follow-up. Autoantibodies have also been used as probes to gain insights into cell biology, helping to isolate and purify intracellular proteins involved in key cellular functions. We give detailed information on two of the most useful techniques for the detection of autoantibodies in the clinical and research laboratory settings, indirect immunofluorescence and immunoblotting. We also discuss several of the antigen-autoantibody systems found in systemic lupus erythematosus (Smith antigen, U1-nuclear ribonucleoprotein, SS-A/Ro, SS-B/La, proliferating cell nuclear antigen ribosomal ribonucleoprotein, double-strand DNA, histones, antiphospholipids, Ku, Ki/SL), systemic sclerosis (centromere, topo I, RNA polymerases, fibrillarin, polymyositis-Scl, Th/To), polymyositis/dermatomyositis (transferRNA synthetases, signal recognition particle, and others), and SS (SS-A/Ro, SS-B/La, nucleolar organizing region-90, p80-coilin), addressing their clinical significance, common detection methods, immunogenetic associations, and the molecular and cellular biology of the cognate antigens.