The polymerase chain reaction (PCR) has been used previously for the detection and typing of adenoviruses directly in clinical samples. Since under clinical conditions subgenus-specific identification is often sufficient, we extended the genus- and type-specific PCR by a subgenus-specific PCR. By sequencing several loop I4 gene regions of the hexon (major adenovirus coat protein) and comparing them to published sequences, subgenus-specific sequences were identified in this region. By using primers targeted to this region and to a conserved hexon gene region, a multiplex, nonnested PCR for the detection and subgenus-specific identification of adenoviruses could be established. The six subgenus-specific amplimers are distinguishable by agarose gel electrophoresis, and subsequent restriction analysis is not necessary. The specificity of the subgenus-specific primer pairs was tested on 23 adenovirus prototypes, representing all six subgenera, on 9 subgenus B and D intermediate strains, and on 16 subgenus C genome types. Furthermore, multiplex, subgenus-specific PCR was performed directly with 100 clinical specimens, including stool samples, ocular swabs, and throat swabs. Adenoviruses of all subgenera could be detected. Especially for clinical application, the rapid, one-step differentiation between subgenus D adenoviruses, causing the severe and highly contagious epidemic keratoconjunctivitis, and subgenus B and E adenoviruses, causing relative harmless ocular infections, is of great importance. The subgenus-specific PCR could also facilitate the primary classification of unknown virus isolates.