Diverticula cells of larval, nymphal, and adult Dermacentor andersoni (Stiles) ticks demonstrated a non-infective phase of 7 to 11 days after engorgement of blood containing Powassan virus. Other organs such as salivary glands, Gené's organ glands, and accessory glands supported virus multiplication. This was demonstrated by infectivity titrations and observations of fluorescent foci when the tissues were stained with Powassan virus antiserum conjugated with fluorescein isothiocyanate (FITC). Transstadial transfer of virus was observed through ecdysis of larvae to nymphs and from nymphs to adults. This occurred both after the ticks had fed on hamsters (which produced a substantial viremia) and guinea pigs (which did not become viremic). Virus was detected only in larval gut and invaded nymphal salivary glands during ecdysis. Infection localized in gut and salivary glands of engorged nymphs and adults and was not detected in adult female accessory and Gené's glands until repletion. Adult males maintained high virus titers (106.0 mouse LD50 per milliliter) in salivary glands only. Transovarial transfer was not detected, although infectivity was present on the surface of eggs laid by the mediation of an infected Gené's organ gland. Powassan virus was detected in salivary gland secretions, and transmission of virus to hamsters and guinea pigs was demonstrated by the bite of D. andersoni nymphs and adults which were infected by ingestion of infective blood by their antecedent larvae.