Antiperinuclear factor is as sensitive as (0.75 to 0.80) and more specific than (0.94 to 0.97) rheumatoid factor for the diagnosis of rheumatoid arthritis. Although three groups found similar performance characteristics using the assay technique described by Youinou, lower sensitivities have also been reported. To clarify these discrepancies, we investigated each parameter of the assay, including storage time of the oral mucosa cells used as the substrate. Even when the slides were frozen, titers fell by one dilution within the first week and by two dilutions within two weeks after sampling. This decline seemed related to storage rather than to freezing: slides kept at 4 degrees C yielded an apparent three-dilution fall in titers after one week and were unevaluable after longer storage times. Successive freeze-thaw cycles did not influence results when the assay was done on the day the cells were sampled and fixed. Titers in sera stored at -25 degrees C remained unchanged or decreased by no more than one dilution during the first 18 months but declined thereafter. These data emphasize the need for performing the assay on the same day or, at the latest, on the day after fixation of the slides. That this precaution was taken should be specified in the "Methods" section of articles on antiperinuclear factor detection.