Prostatic fluid is unsuitable for preserving dog semen at 4 degrees C and exerts harmful effects upon the spermatozoa during the freezing process. Centrifugation immediately after sperm collection is a common method to remove prostatic admixture. In the present study, dog semen, diluted to 25 x 10(6)/ml, was exposed for 5 min to four different centrifugation speeds (180 x g, 720 x g, 1620 x g and 2880 x g) to determine subsequent sperm losses in the supernatant and to assess sperm survival over time. Using 180 x g as centrifugation speed, 8.9% of the sperm cells was lost upon supematant removal. Using 720 x g, 1620 x g or 2880 x g, sperm losses were lower, 2.3, 0.4 and 0.006%, respectively. After centrifugation, the sperm pellet was rediluted in egg-yolk-Tris extender, cooled and stored for 3 days at 4 degrees C. Motility, progressive motility, membrane integrity and sperm morphology were assessed daily. Acrosomal status was assessed after 3 days of storage. The only functional parameter which was influenced by centrifugation speed was membrane integrity as evaluated by means of SYBR14-PI staining: significantly more dead and moribund sperm cells were found after centrifugation at 1620 x g and 2880 x g after 48 and 72 h of storage at 4 degrees C. When higher initial sperm concentrations (50 x 10(6), 75 x 10(6) or 100 x 10(6)/ml) were evaluated for sperm losses, less than 2.3% of the initial total sperm cells was lost at lower centrifugation speeds. We conclude that centrifuging dog sperm for 5 min at 720 x g is the best strategy to remove prostatic fluid because the loss of sperm cells is acceptable and the functional parameters of the spermatozoa are well preserved, even after 3 days of storage.