Samples of 39 canine mammary gland tumours (MGTs) were examined immunohistochemically for oestrogen receptor (ER-alpha), Bcl-2 protein and Ki67 antigen, and by TUNEL assay for apoptosis. ER-alpha was expressed by 80% (31/39) of the tumours, including all of the 15 benign tumours and 67% (16/42) of the malignant tumours. ER-alpha expression was greater in the benign than in the malignant tumours (P<0.01). Bcl-2 protein was detected in 62% (24/39) of the MGTs, of which 67% (10/15) were benign and 58% (14/24) malignant. No significant difference in Bcl-2 expression between benign and malignant tumours was detected. The Ki67 and TUNEL indices were greater in malignant than in benign tumours (P<0.01). Correlation analysis suggested that ER-alpha and Bcl-2 expression were related, but this observation lacked statistical significance. The levels of cell proliferation and apoptosis did not appear to be significantly correlated with the expression of Bcl-2. A positive relationship was apparent between cell proliferation and apoptosis, whilst a negative correlation between ER-alpha and cell proliferation was demonstrated. In conclusion, the suggestion of a positive correlation between ER-alpha and Bcl-2 in canine MGTs indicates that ER may be the regulator of Bcl-2 protein, as in human breast cancer. In contrast to cell proliferation and apoptosis, ER-alpha and Bcl-2 expression were greater in benign MGTs than in their malignant counterparts.