A novel nested quantitative-competitive polymerase chain reaction (nQC-PCR) assay was developed to quantify as few as ten copies per tube of human cytomegalovirus DNA with an overall dynamic range of 10-10(5) copies per tube. This nQC-PCR assay is based on co-amplification of a mimic DNA and it was evaluated with 26 cerebrospinal fluid (CSF) specimens and 44 serum specimens from 70 CMV-infected AIDS patients, 35 of them were diagnosed of CMV retinitis. An excellent correlation was found between nQC-PCR assay and the commercially available Cobas Amplicor CMV Monitor trade mark (CACM) assay (R = 0.9999; P < 0.001; n = 42). Moreover, 13 serum samples with CMV viral loads undetectable with the CACM were successfully quantified by nQC-PCR. CMV viral load was significantly higher in patients with CMV retinitis (P = 0.003). The nQC-PCR assay described below is a very sensitive test for accurate quantitative detection of CMV DNA in different clinical specimens that avoids the need for high-cost instrumentation.