The experiment was conducted to investigate the effectiveness of milking stimulus on oxytocin release and to compare the effect of milking frequency on plasma levels of oxytocin and milk parameters. Twelve Lacaune ewes were subjected to six treatments (T1, T2, T3, T4, T5 and T7 daily milkings) during 6 days. At each milking, blood was sampled and plasma oxytocin levels were determined by enzyme immunoassay. Baseline levels of oxytocin were similar for all milking frequencies. The start of milking was followed by a significant increase in oxytocin levels for all milking frequencies. One daily milking induced significantly higher oxytocin levels than 2, 3, 4, 5 and 7 daily milkings. Milk yield was significantly increased between 4 (1787.0+/-141.5ml) and 7 (1780.0+/-53.6ml) daily milkings compared to 1 (1104.0+/-81.2ml) daily milking. Total concentration of milk protein did not change, but the total milk fat yield for 5 (73.0+/-2.0g/l) and 7 (72.8+/-1.4g/l) daily milkings were significantly higher than for 1 (58.1+/-4.3g/l) daily milking. This study confirmed milk yield gains caused by frequent milk ejection and also showed that oxytocin release was not a limiting factor for milk yield gain when daily milking frequency was increased.