This study investigated the effects of separating dairy calves from their mothers at 1 day (early separation) and 14 days (late separation) after birth. Behavioural observations were conducted on 24 Holstein dairy cow-calf pairs during the first 24h after separation. Before separation, cow-calf pairs were generally inactive. After separation, cows from the late-separation treatment group showed higher rates of calling, movement and placing the head outside the pen, than cows in the early-separation group. Parity did not influence cow behaviour. During the first 2 weeks after calving, cows in the late-separation group (i.e. still with their calves) yielded less milk at milking, a difference at least partly due to the milk consumed by the calf. Milk yields from days 15-150 did not differ between the two groups. After separation, calves in the late-separation group moved and placed their heads outside the pen more often than early-separation calves. During the first 14 days after birth, late-separation calves gained weight at more than three times the rate of those separated early. When introduced to an unfamiliar calf at 6 weeks of age, calves from the late-separation group showed more intense social behaviour towards the unfamiliar calf than did those calves separated early. Thus, the response to separation by both cows and calves increased when calves were separated at 2 weeks rather than 1 day of age, but calves separated at the later age gained more weight and delayed separation appeared to influence the development of calf social behaviour.