Vague and conflicting suggestions currently exist within the literature regarding the appropriate level of ambient illumination to use while conducting the clinical duochrome (bichrome) test. One proposal is that reduced illumination should be adopted since this allows the pupils to dilate, thereby increasing the chromatic interval and resultant test sensitivity. The aim of the present study was to determine whether changes in pupil size and ambient illumination do indeed alter the subjective duochrome response. Accordingly, eight young, visually normal subjects were dilated with 2.5 percent phenylephrine and tested while monocularly viewing the duochrome chart through 1, 3, 5 and 7mm artificial pupils. Additionally, the effects of performing the test through a 1.0 log unit neutral density filter and varying the overall room illumination were examined. There were no significant differences between the mean responses under all test conditions. The variability of individual responses decreased with increasing pupil size; however, a 4mm change in pupil diameter was required to alter the response variability significantly. Therefore it is recommended that the test be performed under the minimum practical ambient illumination conditions in order to minimize this variability.