We investigated the effect of the presence and absence of lambs and suckling by lambs to attenuate activation of the hypothalamo-pituitary adrenal (HPA) axis to isolation and restraint stress in lactating sheep. In Experiment 1, blood samples were collected every 10 min from non-lactating (n = 5) and lactating (n = 5) ewes for 4 h before and during stress. In Experiment 2, ewes (n = 6) were allocated to: 1) non-lactating, 2) lactating with lambs absent, 3) lactating with lambs present but unable to suckle and 4) lactating with lambs present and able to suckle. Blood samples were collected over 8 h with no stress (Control Day) and for 4 h before and 4 h during stress (Stress Day). In Experiment 1, the mean (±SEM) cortisol concentrations increased significantly (P < 0.05) in non-lactating ewes during stress but did not change in lactating ewes. In Experiment 2, cortisol did not vary on the Control Day or pre-treatment of the Stress Day but increased (P < 0.05) during stress in all groups except lactating ewes with lambs present and able to suckle. The greatest cortisol response occurred in non-lactating ewes followed by lactating ewes with lambs absent and lactating ewes with lambs present but unable to suckle. During stress, the ACTH concentrations increased (P < 0.05) in non-lactating ewes and lactating ewes with lambs absent but not in lactating ewes with lambs present. We conclude that the activity of the HPA axis during isolation and restraint is reduced in lactating ewes and that the presence of lambs increases this level of attenuation.