Abstract Adeno-associated viruses (AAV) are defective parvoviruses which require the presence of a helper virus, either an adenovirus or a herpesvirus, to initiate their replication cycle. The mechanisms by which these helper viruses can promote AAV macromolecular synthesis have been investigated in systems where the replication of the helper virus was altered. In the first system, phosphonoacetic acid (PAA), a specific inhibitor of herpesvirus-coded DNA polymerase, was used in AAV-herpes simplex virus (HSV) coinfections to determine what effect this drug would have on the replication of AAV. It was found that in the presence of increasing concentrations of PAA, the synthesis of AAV DNA, structural proteins, and immunofluorescent (IF) capsid antigens was inhibited. However, when an adenovirus helper was used in place of HSV, this inhibition was not seen. It was also found that the addition of the drug 5 hr after infection was still effective in inhibiting AAV capsid antigen synthesis completely. This finding indicates that the PAA-sensitive event required by AAV occurs relatively late in the HSV cycle. Restoration of HSV DNA polymerase activity by reversal of a PAA block was not sufficient for initiating AAV replication. De novo protein synthesis was required also. In the second system, the effects of 2-deoxy- d-glucose (2DG), an inhibitor of protein glycosylation, on AAV replication were also examined. In AAV coinfections with HSV, increasing concentrations of 2DG inhibited the production of AAV IF capsid antigens. Conversely, production of AAV intracellular proteins was enhanced with increasing 2DG concentrations. Under these conditions the pattern of IF staining for the major AAV polypeptide was normal. Thus 2DG appears to interfere with the assembly of AAV proteins into a capsid configuration. In experiments with an adenovirus helper, 2DG was found to inhibit initiation of AAV replication through early adenovirus functions.