The non-stimulated and phorbol 12-myristate 13-acetate (PMA)-stimulated luminol-augmented cellular chemiluminescence (CL) response and viability of milk and blood polymorphonuclear leukocytes (PMN) were determined in lactating dairy cows during different stages of lactation. In the first study, ten healthy cows each in early, mid and late lactation were compared. In a second study, the same measurements as in the first study were evaluated longitudinally in 12 cows during 1 month following parturition. The CL activity and myeloperoxidase (MPO) content of milk PMN and macrophages (M) were also compared. Milk M did not possess MPO activity and were devoid of any luminol-enhanced CL. The CL activity of milk and blood PMN was significantly lower in early lactation than in mid and late lactation (P < 0·001). Whereas little changes were observed in viability of blood PMN, the viability of milk PMN was lower in early lactation than in mid and late lactation (P < 0·001). The percentage of PMN in isolated milk cells was also lower during early lactation than during mid and late lactation (P < 0·001). The CL activity in response to PMA during early, mid and late lactation increased 13, 59 and 42-fold in blood PMN and 1·7, 2·6 and 2·4-fold in milk PMN, respectively, in comparison with non-stimulated PMN. The CL activity, both in milk and blood PMN, the milk PMN viability and the percentage of milk PMN were lowest between 3 d and 11 d post partum. These observed changes immediately after calving could contribute to a higher susceptibility to mastitis in that period.