The internal arrangement of the mammary gland cavity system, cisternal and alveolar milk fractions and the characteristics of milk ejection were investigated in buffaloes. Twenty-four Murrah buffaloes in three different stages of lactation and of two age groups were used. Continuous ultrasound cross-sections during milk ejection induced by exogenous oxytocin were performed to record the latency period of milk ejection. Buffaloes had small cisterns and the cavity area in the teat and gland regions were not significantly different (P>0·05). The animals had long teat canals (3·1±0·1 cm), longer in the hind than fore quarters. Cisternal milk yield was low (0·17±0·01 kg) and cisternal fraction was only 4·9±0·1% of the total milk. The cisternal area (cm2) was 69·6±4·6, 51·61±4·8 and 26·01±4·8 while the cisternal yield (kg) was 0·32±0·05, 0·18±0·05 and 0·05±0·05 in early, mid and late lactation, respectively. A close correlation (r=0·87, P<0·05) existed between the ultrasound cisternal area and cisternal milk yield. The latency period of induced milk ejection was similar to that reported for cows (25±1 s) and was negatively correlated with milk yield (r=−0·75, P<0·05). Milk ejection occurred shortly after elevated oxytocin concentrations were present. Delayed milk ejection reported earlier in this species must therefore be due to the absence of cisternal milk and delayed oxytocin release. An increase in teat length and circumference at milk ejection was also evident in the ultrasound cross sections.