The aim of this study was to investigate possible interactions between the analgesic activity of ketamine (an N-methyl-D-aspartate antagonist), midazolam (a benzodiazepine derivative) and morphine using the tail-flick test in rats. Animals were treated s.c. with ketamine (1.0-10.0 mg/kg), midazolam (0.3 mg/kg), or morphine (0.6 mg/kg) alone. or in combination The strongest analgesic effect of ketamine was observed after 3.0 mg/kg. In higher doses no enhancement of ketamine activity were found. After morphine and ketamine (3.0 mg/kg) or morphine, midazolam and ketamine co-administration. higher antinociceptive effects compared to ketamine activity were found. Rats administered midazolam and ketamine (3.0 mg/kg) showed a decrease of the effect of ketamine analgesia, and the antinociceptive effect of the three-component mixture was lower than after co-injection of morphine and ketamine. The interaction of these two compounds with ketamine (5.0 mg/kg) occurred in a different manner, because midazolam led to a strong enhancement of ketamine analgesia. After morphine and ketamine (5.0 mg/kg) administration, very weak increase of ketamine analgesia was observed. The results of this study allow better understanding of the alteration of the analgesic effects of low doses of ketamine under the influence of morphine and midazolam.