Anti-Ro (SS-A) antibodies are important diagnostic markers for primary Sjögren's syndrome and systemic lupus erythematosus, but their detection by indirect immunofluorescence (IF) in the diagnostic laboratory is hindered by the low cellular abundance of 60kDa Ro protein (Ro60). The approach we used to overcome this problem was to transfect and over-express the Ro60 gene into HEp-2 cells. In this study we have used a mixture of Ro60 transfectants and untransfected HEp-2 cells (HEp-Ro60) as a substrate for IF-antinuclear antibody (ANA) testing in a hospital laboratory. Screening of 240 routine serum specimens identified 14 Ro transfectant-positive sera which were confirmed by counterimmunoelectrophoresis (CIE); 3 of these sera were ANA-negative on untransfected cells and regular HEp-2. A comparison of HEp-Ro60 and regular HEp-2 showed strong concordance of the different ANA patterns between the 2 substrates. No increase in background staining was observed on the Ro transfectants when reacted with normal human sera. A comparison between HEp-Ro60 and CIE for 53 sera from patients with primary Sjögren's syndrome showed that HEp-Ro60 were a sensitive and specific substrate for detection of anti-Ro antibodies. Masking of positive Ro transfectants was observed rarely in sera containing multiple ANA specificities, but the Ro60 staining on these transfectants were unmasked at higher serum dilutions. We conclude that HEp-Ro60 are a suitable substrate for IF-ANA in the routine laboratory and that they have the additional advantage over regular HEp-2 slides of being able to detect anti-Ro in ANA-negative sera. HEp-RO60 are also a valuable confirmatory test for sera giving equivocal precipitin reactions or ELISA results.