Abstract A temperature-sensitive mutant ( tsA13) of human adenovirus type 31 (H31), defective in viral DNA replication, was able to support growth of adeno-associated virus type 1 (AAV1) at the nonpermissive temperature (40°). With the use of this system, the multiplication of AAV1 and AAV1-specific changes were investigated. The latent period of AAV1 growth was shortened by preinfection of cells with H31 tsA13 10 hr before superinfection with AAV1. The rate of DNA synthesis began to rise at about 6 hr postinfection (p.i.) with AAV1 and reached its maximum at 16 hr p.i. In cells coinfected with H31 tsA13 and AAV1, only AAV1 DNA was detected without the presence of adenovirus DNA at 40°. Replicative intermediates of AAV1 DNA were larger than AAV1 DNA in neutral and alkaline sucrose gradients. Specific inclusions induced by AAV1 were observed in the nucleus of coinfected and stained cells. Microscopic autoradiogram of coinfected cells revealed that grains (viral DNA) were found before the appearance of the inclusions of AAV1 in the interior of the nucleus. The AAV1 virion antigen first appeared in the nucleus at about 6 hr p.i. with AAV1 and spread into the cytoplasm within 12 hr p.i. The electron microscopic examination of infected cells revealed that the inclusions were aggregates or crystalline arrays of AAV1 particles in the nucleus. However, neither adenovirus inclusions nor particles were observed. AAV1 DNA replication proceeded in the presence of cycloheximide. The time interval between AAV1 infection and the peak of DNA synthesis became shorter, when AAV1 was superinfected at 16 hr or later after infection with H31 tsA13. These observations suggest that AAV1 lacks its own early protein, and that the lack of the early protein is complemented by a factor(s) induced in adenovirus-infected cells.