Historical survey data suggest that the seroprevalence of antibodies against Coxiella burnetii in the general population of The Netherlands decreased from more than 40% in 1983 to 2.4% in 2007, just before the start of the large 2007-2010 Q fever epidemic. To assess whether the sharp decline in seroprevalence was real, we performed a cross-sectional study using historical samples. We tested samples using a contemporary commercial indirect immunofluorescence assay. In plasma samples from the south of The Netherlands from 1987, we found an age- and sex-standardized seroprevalence of 14.4% (95% confidence interval 11.2-18.3). This was significantly lower than a 1983 estimate from the same area (62.5%), but significantly higher than 2008 (1.0%) and 2010 (2.3%) estimates from the same area. The study suggests that there was a steady and sharp decline in Q fever seroprevalence in the south of The Netherlands from 1987 to 2008. We assume that seroprevalence has decreased in other parts of The Netherlands as well and seroprevalence surveys in other European countries have shown a similar declining trend. Waning population immunity in The Netherlands may have contributed to the scale of the 2007-2010 Q fever epidemic. For a better understanding of the infection dynamics of Q fever, we advocate an international comparative study of the seroprevalence of C. burnetii.