Progesterone and estradiol play a crucial role in the control of mammary gland proliferation and tumour formation in the dog. However, little is known whether steroid metabolizing enzymes are present within the canine mammary gland that may play a modulating role in the bioavailability of progesterone and estrogen. In this study we investigated the expression of the steroid metabolizing enzymes 5alpha-reductase (type I and type II) and aromatase in relation to hyperplasia or tumorigenesis in the canine mammary tissue. The relative mRNA concentrations were examined by a semi-quantitative reverse-transcriptase PCR analysis (RT-PCR). In addition the affinity of dihydroprogesterone (5alpha-reduced metabolite of progesterone) for canine progesterone receptors was investigated. Quantification of the RT-PCR products revealed that in mammary tumours a significantly higher expression of aromatase is present in comparison to normal mammary tissue. Furthermore, significant decrease in expression of both aromatase and 5alpha-reductase type II enzymes was found in hyperplasic mammary tissue compared to tumours. The changes in expression of type II 5alpha-reductase and aromatase were highly correlated. 5alpha-Reduction of progesterone to dihydroprogesterone resulted in a six-fold less affinity for the canine progesterone receptor. It is concluded that hyperplasia is associated with a decreased expression of type II 5alpha-reductase and aromatase enzymes, whereas in tumours the opposite situation is found.