Despite considerable cryobiology research there is no industry standard for the concentration to which ram spermatozoa should be diluted before freezing. Ram semen is highly concentrated and often frozen at a high sperm concentration, necessitating the use of small laparoscopic insemination doses. The aim of this paper was to ascertain the effect of dilution on the integrity of frozen-thawed ram spermatozoa. In the first experiment, spermatozoa were extended with a Tris-buffered diluent before freezing or after thawing to yield a final sperm concentration of 20 x 10(6)/ml, or were not diluted. Motility characteristics, viability and acrosome integrity of spermatozoa were analysed over a 6h incubation period at 37 degrees C. In the second experiment, spermatozoa were either diluted before freezing, subjected to sex-sorting or not diluted before freezing. Thawed spermatozoa were separated into sub-populations using centrifugal counter-current distribution (CCCD) and the profile of partition and functional integrity (viability, chlortetracycline status and Annexin-V binding) in the sub-populations assessed. Dilution before freezing significantly improved post-thaw viability, acrosome integrity and total motility whereas dilution post-thaw decreased viability and motility of spermatozoa. Sperm heterogeneity, as assessed by CCCD profile, was not different for control, diluted and sex-sorted spermatozoa. Analysis of CCCD sub-populations showed the proportion of functional cells (displaying the F-Pattern or no PS translocation) was similar for all sperm types. The results show that ram spermatozoa retain normal function at higher pre-freeze dilution rates than are commonly used in the sheep industry. The application of these findings would result in more practicable and functional artificial insemination doses.