An experiment was performed in primiparous dairy cows (n = 23) to examine consistency of individual differences in reactivity to milking, and correlations between measures of behavior, physiology, and milk ejection. Responsiveness to milking was monitored during the first machine milking, on d 2 of lactation, and during milkings on d 4 and 130 of lactation. Measurements included kicking and stepping behavior, plasma cortisol and plasma oxytocin, heart rate, milk yield, milking time, milk flow rate, and residual milk obtained after administration of exogenous oxytocin. With repeated early lactation milkings, residual milk and the incidence of abnormal milk flow curves decreased. On d 130 of lactation all heifers exhibited normal milk ejection. Except for higher plasma cortisol concentrations on d 2, all measures were consistent over time between d 2 and 4 of lactation as indicated by significant rank correlations. Individual differences in the behavioral response to udder preparation were consistent over time between early lactation milkings and d 130 of lactation. Residual milk, milk yield, maximum milk flow rate, plasma oxytocin and heart rate during udder preparation were similarly interrelated on d 2 and 4 of lactation. High heart rate responses on d 2 and 4 were associated with enhanced inhibition of milk ejection. In contrast, behavior recorded during the milking process was unrelated to ease of milk removal. Our results indicate that milking at the beginning of lactation may be stressful to some heifers, to the extent that milk ejection is inhibited, but less disturbing to others. The existence of consistent behavioral and physiological responses in the present study suggests that responsiveness of dairy heifers to milking is mediated by stable animal characteristics.