Published information on contrast detection threshold is based primarily on research using a location-known methodology. In previous work on testing the Digital Imaging and Communications in Medicine (DICOM) Grayscale Standard Display Function (GSDF) for perceptual linearity, this research group used a location-unknown methodology to more closely reflect clinical practice. A high false-positive rate resulted in a high variance leading to the conclusion that the impact on results of employing a location-known methodology needed to be explored. Fourteen readers reviewed two sets of simulated mammographic background images, one with the location-unknown and one with the location-known methodology. The results of the reader study were analyzed using Reader Operating Characteristic (ROC) methodology and a paired t test. Contrast detection threshold was analyzed using contingency tables. No statistically significant difference was found in GSDF testing, but a highly statistical significant difference (p value <0.0001) was seen in the ROC (AUC) curve between the location-unknown and the location-known methodologies. Location-known methodology not only improved the power of the GSDF test but also affected the contrast detection threshold which changed from +3 when the location was unknown to +2 gray levels for the location-known images. The selection of location known versus unknown in experimental design must be carefully considered to ensure that the conclusions of the experiment reflect the study's objectives.