Standardization of the indirect immunofluorescence antinuclear antibody (IF-ANA) test can be improved for a given substrate with use of reference ANA sera, uniform assay conditions, and standardization of optical systems. To accomplish this, reference sera from the Arthritis Foundation with defined antibody specificities for nDNA, SS-B, RNP, Sm, nucleoli, and "speckled pattern" were reacted with commonly used IF-ANA substrates, mouse kidney sections, KB and HEp-2 tissue culture cells. Reagents and assay conditions used were those provided with the substrates in commercially available IF-ANA kits. A microscope slide with graded intensities of fluorescent beads was used to standardize microscope fluorescence intensity readings. The authors' fluorescence pattern and intensity results should be directly comparable to results obtained in other laboratories for the six antibodies for which reference sera are available. Although no defined sera are widely available for SS-A, Scl-70, PM-1, and centromere antibodies, the ability of each substrate to detect these antinuclear antibodies as well as mitochondrial, smooth muscle, ribosomal and microsomal antibodies also was tested. HEp-2 cells and KB cells were found to be superior to mouse kidney sections for detection of SS-A, Scl-70, PM-1 and centromere antinuclear antibodies. Mouse kidney sections were superior for screening of sera for the absence of ANA as well as for detection of smooth muscle and liver-kidney microsomal antibodies. Other antibodies were detected with equal sensitivity with all substrates and each of the three ANA kits used in the study performed satisfactorily. Use of reference sera as well as optical standardization is recommended for IF-ANA testing.