The aim of the present study was to assess the effect of the addition of docosahexaenoic acid (DHA) on the in vitro quality of cooled and frozen-thawed stallion semen. In Experiment 1, semen from 10 stallions was collected (three ejaculates per stallion). Semen was diluted to 100 x 10(6) spermatozoa mL(-1) with 0.02 mM vitamin E (VE) and 0, 1, 10 or 20 ng mL(-1) DHA and frozen. Semen was thawed and total motility (TM), rapid progressive motility (PM), acrosome integrity, membrane fluidity and morphology were assessed. In Experiment 2, semen from three stallions was collected (three ejaculates per stallion) and frozen as in Experiment 1, but VE and DHA were added after thawing. TM and PM were assessed at 30, 60 and 120 min and viability, acrosome integrity and membrane fluidity were evaluated at 30 min. In Experiment 3, semen from five stallions was collected (one to three ejaculates per stallion), diluted to 20 x 10(6) spermatozoamL(-1) and stored at 4 degrees C. After 1, 24, 48 and 72 h, TM, PM, viability, membrane fluidity and lipid peroxidation were assessed. The addition of DHA had no effect on frozen semen (Experiments 1 and 2) but improved TM, PM and membrane fluidity in cooled stallion semen.