We studied 35 patients, in whom infection with herpesviruses had been a differential diagnosis clinically but in whom a biopsy specimen had been taken for confirmation of the diagnosis, because considerable doubts existed about the clinical diagnosis. Only those patients were included in the study in whom sections cut from the biopsy specimen had been interpreted as being "not unequivocally diagnostic" of herpesvirus infection by two independent dermatopathologists. Findings clinical and histopathologic were correlated with results from PCR studies on the formalin-fixed paraffin embedded tissue. Total genomic and viral DNA was isolated from paraffin-embedded tissue samples (QIAmp DNA Kit; QIAGEN). About 15 sections (5 m) were used for each preparation. HSV-1-, HSV-2-, and VZV- specific PCR fragments were amplified (HSV-1: genomic region amplified: RL 2; primers H1P32/H1M32, HSV-2: genomic region amplified: UL 28; primers H2M40/H2P4, VZV: genomic region amplified: ORF 8, primers VP22/VM20).[4] PCR reactions were performed in 12.5-l reaction mixtures (1x reaction buffer, HotStarTaq QUIAGEN; 3.5 mM MgCl2; 1.2 M of each primer; about 40–60 ng total DNA; 200 M of each dNTP; 2.5 units HotStarTaq DNA polymerase QIAGEN). The thermal profile was: 1. 95°C/15 min (1 cycle); 2. 94°C/20 sec ; 60°C/30 sec ; 72°C/30 sec (30 cycles) ; 3. 72°C/5 min (1 cycle); 4. 94°C/20 sec; 58°C/1min; 72°C/30 sec (20 cycles) ; 72°C/15 min (1 cycle). PCR products were analyzed subsequently in 2% agarose gels. To identify unambiguously all three virus types, some PCR products were also used directly in dideoxy-termination sequencing reactions (Big Dye Terminator Cycle Sequencing Mix; Applied Bio-Systems). Cycle sequencing reactions were performed in 20-l reaction volumes (3 l Big Dye; 5 l 2.5x sequencing buffer, Applied Bio-Systems; 10 ml of the amplified products [200–350 ng]; 2 l primer [1 M]. The thermal profile was: 96°C/30 sec; 55°C/15 sec; 60°C/4 min (30 cycles). After removal of unincorporated dye terminators (Dye Ex, QIAGEN) sequencing reactions were denaturated and the samples were run on an ABI Prism 377 automated sequencer (Applied Bio-Systems). Sequences were further processed using Bio Edit version 5.0.9 [5] and subsequently analyzed using the NCBI BLAST search.