The fiercest challenge yet both to Pericles' leadership and to the unity of the Delian League arose in 441 from a dispute with the island city-state of Samos, one of the few alliance members still fielding a strong navy of its own. The Samians and their neighbors on the mainland in the city-state of Miletus, also a league member, had reached an impasse in a heated conflict to take over some nearby territory. The Athenians instructed their arguing allies to go to arbitration, but Samos refused. Pericles probably proposed the decision made by Athens as leader of the Delian League to respond very forcibly by installing a new, and democratic, government on Samos; in supporting this policy unfavorable to the elite of Samos, Pericles was perhaps recalling the treachery of those Samian commanders fifty years earlier who had made a deal with the Persian king Darius to save their city-state from what they feared would be devastation and therefore deserted the Ionian alliance in the heat of the Battle of Lade. A fine of 80 talents was imposed, and 100 citizens were taken away as diplomatic hostages to an Athenian stronghold on the island of Lemnos. In keeping with his conspicuous incorruptibility, Pericles refused a king's ransom in bribes offered him by a group of rich antidemocratic Samians and the Persian governor to ease the conditions.

The next year, the dispute blew up into a crisis that restored the Persians to the status of an active threat to the member states of the Delian League when the Samian rebels allied with the Persian governor in western Anatolia. Now flush with money supplied by the Persian king's governor, they hired mercenaries and violently overthrew the newly installed democracy allied with Athens. The anti-Athenian Samians also freed the hostages and captured some Athenian citizens. These fellow Greeks they turned over to their Persian backer. The city-state of Byzantium then joined the rebellious Samians in rebelling against the league.

To meet this serious crisis to the league's unity and to the safety of his fellow citizens who were being held as hostages, Pericles was tasked with commanding a force of warships from Athens supported by the fleets of Chios and Lesbos.