Streptococcus uberis causes a significant proportion of clinical and subclinical intramammary infections (IMI) in lactating and non-lactating dairy cows. In spite of this, its pathogenesis is incompletely understood. A study was conducted to determine leukocyte and cytokine dynamics during experimentally induced S. uberis mastitis. Five Jersey and five Holstein cows were challenged via intramammary inoculation of S. uberis into two uninfected mammary glands. Sixteen of 20 challenged mammary glands developed clinical mastitis with peak clinical signs observed at 144 h. The number of S. uberis in milk increased (P<0.05) 48 h after challenge, in spite of an increase in milk somatic cells that began at 18 h (P<0.001) and remained elevated throughout the study. Increased tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-8 (IL-8) in milk were detected 66 h after challenge (P<0.05). Peak TNF-alpha and IL-8 concentrations occurred 120 h after challenge and preceded peak clinical signs. Experimental S. uberis IMI induced local production of TNF-alpha, IL-1beta and IL-8, which may play a role in the pathogenesis of S. uberis mastitis. Other mediators may be involved in initial leukocyte recruitment to the mammary gland, since increases in milk somatic cells occurred earlier than cytokine production.