Although human infection with adenovirus-associated virus (AAV) has been demonstrated, there is no evidence that disease results from such infections. The proportion of adenovirus infections which are dual infections with AAV is virtually unknown, since special methods are required to demonstrate infection with AAV. To search for AAV, we re-examined a collection of specimens from 40 persons involved in an epidemic of pharyngoconjunctival fever associated with a swimming pool. Virological and serological studies indicated that the etiological agent was adenovirus type 3. When the 91 original eye, throat, and fecal specimens were re-examined, using methods suitable for detection of adenovirus and AAV, 37 strains of adenovirus type 3 and 35 strains of AAV type 3 (AAV3) were isolated. Surprisingly, 19 AAV3 but only 11 adenovirus isolates were found in eye specimens, whereas adenovirus isolates were equally distributed in all types of specimens. Four AAV3 strains were isolated from adults. Significant (fourfold or greater) rises in AAV3 complement-fixing antibody titers were seen in six of 14 persons shedding AAV3, whereas nine of 10 persons shedding adenovirus type 3 showed significant rises in adenovirus complement-fixing antibody. These results raise the question whether AAV persists better in eyes than adenovirus or that a possible association with conjunctivitis might be present. In contrast to the results in the specimens from the swimming pool epidemic, only one of 36 adenovirus strains isolated in other Seattle-based studies yielded AAV. Complement fixation tests on serial sets of sera collected from 60 children not involved in the swimming pool episode revealed nine AAV2 and 12 AAV3 infections during a 4-year period.