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To evaluate the prevalence and the dynamics of HIV-1 subtypes in Romanian adults and children, and to investigate the origins of the nosocomial epidemic.A total of 1000 serum and plasma samples, from adults (n = 579) and children (n = 421) who were diagnosed as being HIV-1-infected during 1990–1997 in 39 of the 41 Romanian districts, were serotyped. Viral DNA was isolated from blood samples of 84 patients and the viruses were genotyped.Serotyping was performed with a peptide subtype-specific enzyme immunoassay (SSEIA), based on in vitro competition for antibody binding between the representative V3 peptides of the different clades (A–F). Proviral HIV-1 DNA was genotyped by heteroduplex mobility assay or by sequence analysis of the C2–V3 env region.SSEIA showed that 93% of the samples from horizontally infected children were serotype F, 1% were serotype B, and the remaining 6% were uninterpretable. In vertically infected children, 74% of strains were serotype F, 10% were serotype A, 3% were serotype B, and 3% were serotype E. Serotype F was also the dominant subtype in adults (68%), but serotypes A, B, C, D and E were also detected. SSEIA gave indeterminate results in 7% of cases. A strong correlation (90%) between serotyping and genotyping for subtype F was found. Analysis of the relative incidence of the different serotypes over a 7-year period (1990–1997) showed a stable distribution.Subtype F largely dominates the epidemiology of HIV-1 infection in both children and adults in Romania, although other major subtypes are present. The predominance of subtype F in Romania may be a future potential source of HIV-1 variability in Europe.