The human cytomegalovirus (HCMV) gene UL97 product was shown to play an important role in phosphorylation of ganciclovir (GCV) in HCMV-infected cells. The deletion of the 4 amino-acid sequence AACR confers resistance to a laboratory mutant. The aim of this study was to develop a rapid and simple method to detect mutations within the 12 base pair (bp) fragment encoding AACR, from isolates and clinical specimens (urine, bronchoalveolar lavage, cerebral spinal fluid samples). A target region encompassing this 12bp sequence was amplified by a single-step PCR assay from HCMV isolates and reference strains, and a two-step procedure from clinical specimens. Reaction products were submitted to restriction enzyme analysis and dot-blot hybridization assay. Two biotinylated probes were used: one probe (DL) overlapping the 12bp region; and a control probe with similar length and GC content. Hybridization was performed under conditions allowing the detection of one bp deletion (HCMV strain susceptibility to GCV was determined by a rapid late antigen synthesis reduction assay.) The control probe hybridized to the UL97 sequence amplified from all 23 tested isolates and the reference strains. The DL probe gave a positive signal with GCV-susceptible strains; no signal was obtained for five out of seven resistant isolates, and for a laboratory mutant derived from the strain AD169. Restriction analysis of amplification products showed different patterns suggesting this region can be involved in various DNA changes.(ABSTRACT TRUNCATED AT 250 WORDS)