Sequence analysis revealed phospholipase A sub(2) (PLA sub(2)) motifs in capsid proteins of parvoviruses. Although PLA sub(2) activity is not known to exist in viruses, putative PLA sub(2)s from divergent parvoviruses, human B19, porcine parvovirus, and insect GmDNV (densovirus from Galleria mellonella), can emulate catalytic properties of secreted PLA sub(2). Mutations of critical amino acids strongly reduce both PLA sub(2) activity and, proportionally, viral infectivity, but cell surface attachment, entry, and endocytosis by PLA sub(2)-deficient virions are not affected. PLA sub(2) activity is critical for efficient transfer of the viral genome from late endosomes/lysosomes to the nucleus to initiate replication. These findings offer the prospect of developing PLA sub(2) inhibitors as a new class of antiviral drugs against parvovirus infections and associated diseases.