Ishihara diagnostic plates of the same design from 10 books (five editions, two books from each) were compared to determine the chromatic and diagnostic variability. The responses of 17 red-green (r-g) deficient subjects were recorded and compared. Spectrophotometric analysis was also performed on five of these books. The chromaticity coordinates of the colored dots were plotted in the 1931 International Commission on Illumination (CIE) chromaticity diagram in reference to the protan and deutan confusion lines. The relative lightness of the figure compared to the background was calculated as the Weber contrast according to the Smith-Pokorny fundamentals for both protans and deutans. Significant diagnostic variation was found among the r-g-deficient subjects. The chromaticity coordinates and the relative lightness of the colored dots were also found to have significant variations. We were able to account for many of the diagnostic differences based on the chromaticity and relative lightness variations among the books. Spectrophotometric analysis may provide additional quality control for these plates. (C) 2003 Wiley Periodicals, Inc.