The value of automatic image analysis in the investigation of nucleolus regions (AgNOR) has been examined in tissue sections of 52 malignant and 30 benign breast lesions. Determination of the AgNOR number per cell alone revealed a considerable overlap between benign (range 1.2-3.8) and malignant specimens (range 1.5-16.2). They differed however, highly significantly (P<0.001) in their AgNOR sizes. In benign breast disorders the mean AgNOR area per tumour ranged from 0.22 μm2 to 1.07 μm2 (mean 0.39 μm2), whereas in carcinomas AgNOR sites ranged from 0.05 μm2 to 0.22 μm2 (mean 0.09 μm2). AgNOR counts showed a good correlation with histopathological grade (P<0.05), aneuploidy (P<0.01), proliferation rate as determined by Ki67 immunostaining (P<0.01), as well as oestrogen and progesterone receptor content (P<0.01). Image analysis proved to be advantageous over AgNOR counting alone as it facilitated the standardization of the AgNOR technique itself and thus, significantly improved its diagnostic specifity. © 1990 Springer-Verlag.