Real-time PCR methods have become widely used within the past few years. However, real-time PCR is rarely used to study chronic diseases with low pathogen loads, presumably because of insufficient sensitivity. In this report, we developed an integrated nucleic acid isolation and real-time PCR platform that vastly improved the sensitivity of the quantitative detection of the intracellular bacterium, Chlamydia spp., by fluorescence resonance energy transfer real-time PCR. Determinants of the overall detection sensitivity were analyzed by extracting nucleic acids from bovine milk specimens spiked with low amounts of chlamydial organisms. Nucleic acids were optimally preserved and recovered by collection in guanidinium stabilization buffer, binding to a matrix of glass fiber fleece, and elution in low volume. Step-down thermal cycling and an excess of hot-start Taq polymerase vastly improved the robustness and sensitivity of the real-time PCR while essentially maintaining 100% specificity. The amplification of Chlamydia 23S rRNA allowed for the differentiation of chlamydial species and was more robust at low target numbers than amplification of the omp1 gene. The best combined method detected single targets per a 100-microL specimen equivalent in a 5-microL real-time PCR input. In an initial application, this high-sensitivity real-time PCR platform demonstrated a high prevalence of chlamydial infection in cattle.