Behavioural-antennal responses of tsetse to buffalo urine, which has been identified as a potent olfactory attractant, were investigated. Tsetse Glossina morsitans morsitans rapidly raised their antennae upon stimulation with the odour of buffalo urine. Flies showed a clear preference for the undiluted urine, though antennal responses even to 100,000 × diluted urine were significantly different from control, indicating the high efficacy of the attracting compounds present in it. Fresh buffalo urine was the most attracting as compared to 4- and 8-day-old urine. The dichloromethane extract of fresh urine was as active as the natural product, indicating that the attracting compounds were extractable into dichloromethane. Under laboratory conditions, responses of males to the urine were significantly more than females.