Abstract Background Androgen acts via androgen receptor (AR) and accurate measurement of the levels of AR protein expression is critical for prostate research. The expression of AR in paired specimens of benign prostate and prostate cancer from 20 African and 20 Caucasian Americans was compared to demonstrate an application of this system. Methods A set of 200 immunopositive and 200 immunonegative nuclei were collected from the images using a macro developed in Image Pro Plus. Linear Discriminant and Logistic Regression analyses were performed on the data to generate classification coefficients. Classification coefficients render the automated image analysis software independent of the type of immunostaining or image acquisition system used. The image analysis software performs local segmentation and uses nuclear shape and size to detect prostatic epithelial nuclei. AR expression is described by (a) percentage of immunopositive nuclei; (b) percentage of immunopositive nuclear area; and (c) intensity of AR expression among immunopositive nuclei or areas. Results The percent positive nuclei and percent nuclear area were similar by race in both benign prostate hyperplasia and prostate cancer. In prostate cancer epithelial nuclei, African Americans exhibited 38% higher levels of AR immunostaining than Caucasian Americans (two sided Student's t-tests; P < 0.05). Intensity of AR immunostaining was similar between races in benign prostate. Conclusion The differences measured in the intensity of AR expression in prostate cancer were consistent with previous studies. Classification coefficients are required due to non-standardized immunostaining and image collection methods across medical institutions and research laboratories and helps customize the software for the specimen under study. The availability of a free, automated system creates new opportunities for testing, evaluation and use of this image analysis system by many research groups who study nuclear protein expression.