The aim of this study was to investigate the impact of a 24-h cooling period prior to freezing on domestic cat epididymal sperm viability. Fifteen tomcats were submitted to routine orchiectomy and sperm samples were retrieved from both epididymides in a Tris-glucose-20% egg yolk extender. For each tomcat, the diluted sperm was split into two equal volumes and cooled to 5 degrees C at a rate of 0.5 degrees C/min; one sample for 60 min (control) and the other for 24 h (cooled). After the cooling period, samples from both groups were frozen using an identical freezing protocol. Sperm samples were evaluated in three different periods: immediately after harvesting, after cooling at 5 degrees C for 24 h (cooled group) and after freezing-thawing of control and cooled groups. Evaluations consisted of sperm motility and progressive status, sperm morphology and plasma membrane integrity (PMI) using two fluorescent probes. After cooling for 24 h, a decrease (p < 0.05) in sperm motility, progressive status and PMI was observed when compared to sperm samples immediately after collection. Comparing the results obtained after thawing, no difference (p < 0.05) was found regarding sperm motility, progressive status, PMI and sperm morphology between control and cooled groups. The results from the present study show that cooling cat epididymal spermatozoa at 5 degrees C for 24 h prior to freezing does not lead to major damage of spermatozoa impairing the freeze-thaw process.