Cat semen was diluted at 37°C in Tes-Tris buffer (TesT), pH 7.5, at osmolalities ranging from 195 to 390 m0sm/kg, cooled to 5°C over 90 minutes and stored for 24 hours at that temperature. Motility and percentage of spermatozoa staining with a supravital stain were estimated before cooling, after cooling and after storage for 24 hours. The osmolality of undiluted pooled ejaculates from five animals was measured, and also that of different diluents (citrate with phosphate buffer, lactose and TesT-egg yolk) used for cat semen. The osmolality measurements of cat semen suggested an osmolality of less than 320 m0sm/kg at ejaculation, increasing with time after ejaculation. Varying the egg yolk concentrations (2% to 20%) did not affect the osmolality of TesT diluent. Diluent osmolalities of less than 292 m0sm/kg were found to reduce sperm motility significantly ( P <0.001 ) although there was no significant increase in the percentage of cells staining with a supravital stain, while those greater than 325 m0sm/kg increased the variation of response among animals. Cooling and storage significantly reduced motility ( P <0.01 to P <0.001 ) and increased the number of stained cells ( P <0.001 ). There were significant differences between ejaculates ( P <0.01 ) and significant interactions between osmolality and cooling/storage ( P<0.05 to P<0.001 ). The best overall results were seen with a TesT diluent of 292 to 325 m0sm/kg which supported good motility for at least 24 hours.