We developed a real-time PCR assay for the quantitative detection of Clostridium tyrobutyricum, which has been identified as the major causal agent of late blowing in cheese. The assay was 100% specific, with an analytical sensitivity of 1 genome equivalent in 40% of the reactions. The quantification was linear (R2 > 0.9995) over a 5-log dynamic range, down to 10 genome equivalents, with a PCR efficiency of >0.946. With optimized detergent treatment and enzymatic pretreatment of the sample before centrifugation and nucleic acid extraction, the assay counted down to 300 C. tyrobutyricum spores, with a relative accuracy of 82.98 to 107.68, and detected as few as 25 spores in 25 ml of artificially contaminated raw or ultrahigh-temperature-treated whole milk.This work was supported by the Agrarian Experimental Plan of the ITACyL/Junta de Castilla y León and the European Union's Marie-Curie Mobility Program (contract MEIF-CT-2005-0011564). L.L.-E. received a Ph.D. studentship from the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), D.R.-L. is a fellow of the European Union's Marie-Curie Mobility Program, and M.H. holds a contract from the INIA.Peer reviewed