1. The aim of the present study was to compare ecto-nucleotidase activities in rat bisected vas deferens using 1,N6-etheno(epsilon)-nucleotides (epsilon-ATP and epsilon-AMP) as substrates. Degradation was estimated by measuring the disappearance of the substrate and the appearance of its metabolites using HPLC with fluorescence detection. Incubation of tissue preparations (prostatic or epididymal portions) with 300 nmol/L epsilon-ATP at 37 degrees C caused a partial disappearance of epsilon-ATP and appearance of its metabolites (epsilon-ADP, epsilon-AMP and epsilon-adenosine). Incubation at 25 degrees C reduced epsilon-ATP degradation more in the prostatic than in the epididymal portion. 2. Incubation of tissue preparations with epsilon-AMP at 37 degrees C resulted in the disappearance of epsilon-AMP and the appearance of epsilon-adenosine, which was more pronounced in the epididymal than in the prostatic portion. Incubation at 25 degrees C reduced epsilon-AMP degradation more in the epididymal than in the prostatic portion. 3. Decreasing pH from 7.4 to 6.5 enhanced epsilon-AMP degradation only in the prostatic portion, whereas increasing pH from 7.4 to 8.5 enhanced epsilon-AMP degradation in both portions, but more markedly in the epididymal portion. The alkaline phosphatase inhibitors levamisole (10 mmol/L) and beta-glycerophosphate (10 mmol/L) reduced epsilon-AMP degradation only in the epididymal portion. 4. In conclusion, the results of the present study are compatible with the presence, in the bisected rat vas deferens, of an ecto-nucleotidase system that is involved in the degradation of extracellular purines, which may differ between the epididymal and prostatic portions, with the epididymal portion presenting a different and higher capacity to form adenosine.