A large-scale investigation on Coxiella burnetii was carried out in dairy cattle herds from a Q fever-endemic region to evaluate the degree of exposure to C. burnetii and to estimate prevalences. This study included all of the dairy cattle herds from the province of Bizkaia, Northern Spain (n=178). Herds were visited between September 2009 and February 2010, and 100mL of bulk-tank milk (BTM) per farm was collected to be analyzed by ELISA and PCR. Blood samples were also taken from about 15 animals randomly selected from each herd. One hundred nineteen of the 178 studied herds (66.9±6.9%) were positive for the presence of anti-C. burnetii antibodies in BTM. Serum samples from 1,306 cows, 654 heifers, and 502 calves were analyzed by ELISA, and cows showed a statistically significantly higher seroprevalence (12.3±1.8%) than heifers (1.1±0.8%) and calves (0.0±0.0%). Eighty-nine herds (50.0±7.3%) had at least 1 seropositive animal, but within-herd prevalences higher than 20% were only observed in 24 herds (13.5±5.0%). A significant correlation was observed between BTM ELISA sample-to-positive control ratios and within-herd seroprevalence, being higher when considering only cows (R 2=0.21). Animals from herds with negative BTM by ELISA showed a mean seroprevalence of 2.5%, whereas animals from herds with positive BTM samples had a statistically significantly higher seroprevalence (8.9%, F=19.7, degrees of freedom=1). The proportion of herds C. burnetii positive by BTM PCR was 51.7±7.3% (92/178). The widespread distribution of C. burnetii in cattle advocates for the implementation of Q fever control strategies. ; This work was supported by funding from Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA, Madrid, Spain; RTA 2009-00017-00), European Regional Development Fund (ERDF), and the Department of Agriculture of the Basque Government (Spain). I. Astobiza is the recipient of a predoctoral fellowship from the Department of Agriculture, Fisheries and Food of the Basque Government and A. Piñero is the recipient of a predoctoral fellowship from INIA. ; Peer Reviewed