Successful pre-emptive anti-cytomegalovirus (CMV) therapy relies on sensitive, specific and reproducible tests for CMV detection. Real-time polymerase chain reaction (PCR) for CMV-DNA provides a superior reproducibility and sensitivity than pp65-antigenemia. Evaluation of a novel commercial real-time PCR for CMV-DNA associated with a fully automated DNA extraction from whole blood (WB) was performed, studying the correlation with pp65-antigenemia in guiding pre-emptive therapy. Analytical evaluation showed that PCR correctly quantitated CMV from 500 to 500,000copies/ml with a close correlation with expected values (r=0.999). Clinical evaluation on 375 consecutive WB samples from 48 infected patients (18 pre-emptively treated for pp65 values >/=50 positive cells) showed that according to pp65-antigenemia of 0, 1-10, 11-49 and >/=50 positive cells, median DNA levels were 3.7, 3.9, 4.6 and 5.6 log(10)copies/ml, respectively. According to existing pre-emptive treatment criteria based on pp65-antigenemia, receiver-operating curve analysis indicated 5.3log/ml (200,000genomes/ml) as the best CMV-DNA level to discriminate between patients requiring pre-emptive therapy and those who did not (positive predictive value: 91%; negative predictive value: 74%; sensitivity and specificity: 68 and 93%). In conclusion, real-time PCR provides reliable results for monitoring the developing of CMV infection, allowing for the definition of CMV-DNA thresholds associated with infection progress.