The acid and alkaline endonuclease activities present in bull semen and accessory sex organs were measured by determining the amount of radioactive material solubilized from [3H]DNA gel. The alkaline endonuclease (pH 7.5) required Ca2+ plus Mg2+ for activity, while the acid endonuclease (pH 5.5-6.0) required Ca2+ or Mg2+ for activity. High Ca2+, Mg2+-dependent alkaline endonuclease activity was found in seminal plasma, prostate and seminal vesicle while sperms possessed low activity, suggesting that the seminal plasma enzyme was a secretory product of the accessory sex organs. The highest Mg2+-dependent acid endonuclease activity was found in the cytosol fraction of testis.