The adeno-associated virus (AAV) replication proteins Rep78 and Rep68 regulate viral gene expression and DNA amplification. Their effects on both processes suggest that they play roles in all phases of the virus life cycle. We have investigated Rep protein phosphorylation to determine if this modification might alter Rep function. All four Rep proteins were found to be phosphorylated in AAV and adenovirus co-infected cell cultures, and Rep proteins contained phospho-serine whereas no phospho-threonine or -tyrosine was detected. We also observed that when viral DNA synthesis was inhibited, there was a significant decrease in the level of Rep78/68 phosphorylation. Our results suggest a plausible mechanism whereby AAV Rep 78/68 function may be regulated by phosphorylation.