A DNA amplification test was developed for the sensitive detection of the diarrhoea-associated subgroup F adenoviruses in clinical specimens. The test was made highly specific for serotypes 40 and 41 by using a region of the genome (the long-fiber gene) which is not significantly homologous to other human adenoviruses, but which is highly conserved between Ad40 and Ad41. A positive subgroup F adenovirus diagnosis was characterized by the presence of an amplification product of 152 base pairs, which could be digested into products of predictable length by restriction enzymes XbaI and SpeI. The viruses were typed as either Ad40 or Ad41 by digestion of the amplification product with a restriction enzyme which digested only Ad40 DNA. The specificity of the test was assessed using DNA from other adenoviruses, from human and simian cells, and from bacteria commonly found in the human intestine. There was a strong correlation between results of typing obtained with PCR and restriction enzyme typing of Ad40 and Ad41, and also positivity using subgroup F specific probes in dot blot hybridizations.