Parotid tumor agent was an early name for polyomavirus due to its ability to induce tumors (myoepitheliomas originating from the myoepithelial glandular tissues) in mice inoculated with polyomavirus (Py) as neonates. It has long been thought that these tumors directly follow productive infection by Py in epithelial cells of the salivary gland, allowing subsequent cellular genetic changes leading to tumor formation. Curiously, the ability of salivary glandular tissue to support Py infection has not been experimentally established. Although Southern analysis for Py DNA has shown virus DNA to be present in whole salivary glands during acute infection, salivary glands are composed of various cell types (myoepithelial glandular cells called serous and mucous cells, fibrocollagenous cells, and interstitial cells), not all of which become transformed. We now use in situ hybridization for Py DNA along with immunohistological and immunohistochemical analyses to show that salivary gland serous and mucous cells are nonpermissive for acute Py infection in Balb/C and C3H mice, but are Py infected, as shown by T-ag expression. Salivary gland fibroblasts and interstitial cells, however, were permissive for Py replication. In addition, isoproterenol and tannin, which induce hypertrophy and hyperplasia of the secretory cells of adult male mice salivary glands, did not make these cells permissive to Py replication.