We investigated the differences in the effect of intramammarily infused endotoxin for cows in early (EL) and late (LL) lactation. In this crossover study, nine cows were challenged twice with 100 microg of intramammarily infused Escherichia coli 0111:B4 LPS, each cow serving its own control. Systemic and local signs were recorded throughout the experiment to assess the severity of each cow's response. Daily milk yield and indicators of inflammation in blood and milk were also recorded. The response was significantly more severe in the EL period. Before the challenge, milk yield and serum free fatty acid concentration were higher and serum urea concentration lower in EL compared with LL cows. No significant differences were present in other parameters. After the challenge, milk SCC and leukocyte function, measured as chemiluminescence, increased, more markedly in EL. Blood neutrophil content varied from neutropenia to neutrophilia. Simultaneously with neutropenia, the function of neutrophils increased in LL but decreased in EL. Serum cortisol peaked 4 h after challenge in both groups. In conclusion, endotoxin challenge resulted in more severe response in EL. During that time cows also had impaired neutrophil function postchallenge.