Rapid diagnosis of human herpesvirus primary infections or reactivations is facilitated by quantitative PCRs. Quantitative PCR assays with a standard thermal cycling profile permitting simultaneous detection of herpes simplex virus (HSV), varicella zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV6) DNA were developed and validated for diagnostic use. High specificity and sensitivity were achieved and the new PCR assays correlated well with commercial PCR assays. Twenty two thousand eight hundred sixty eight PCR tests were undertaken on specimens obtained from immunosuppressed patients. DNAemia was frequent with EBV (43.5%), HHV6 (32.4%), CMV (12.8%), and VZV (12.9%). As already described for EBV and CMV, high virus loads of HHV6 and VZV were associated with clinical symptoms and poor clinical outcome, for example, three of four patients with VZV virus loads >10(5) copies/ml died. A high proportion of lower respiratory specimens was positive for EBV- (38.8%), HHV6- (29.4%), and CMV-DNA (18.2%). For CMV, infection was confirmed in 66.7% of patients by virus isolation or positive pp65 antigenaemia. Differentiation of HHV6A, -B and HSV-1, -2 by melting curve analysis revealed that HHV6A and HSV-2 represented only 1.8% and 3.3% of all positive specimens, respectively. In conclusion, these results indicate significant improvements for the early diagnosis of primary infections or reactivations of five human herpesviruses especially in immunosuppressed patients. Detection of coinfections with multiple herpesviruses is facilitated. Quantitative results enable monitoring of virus load during antiviral therapy. A standard thermal cycling profile permits time and cost effective use in a routine diagnostic setting.