Saudi Arabia summoned Iran's ambassador on Tuesday after four Saudis died from poisoning in the northeast Iranian city of Mashhad, Saudi foreign ministry spokesman Osama Naqli said in a statement published on the state news agency.
A group of 33 pilgrims from Saudi Arabia's Shi'ite Muslim minority was exposed to poison on Sunday while staying at a Mashhad hotel, state news agency IRNA reported on Monday.
Another Iranian news agency quoted an official as saying the poisoning appeared to be accidental, blaming poor cleaning standards at the hotel.
Four children died, while another 28 were hospitalised after the incident. Iranian authorities said they arrested five members of hotel staff.
Saudi Arabia's foreign ministry called on Iran to "swiftly take the necessary investigations required and to discover the circumstances surrounding the event, and allow them to follow up on the medical conditions of the patients, and to provide necessary protection for them."
Mashhad is home to the shrine of Imam Reza, a revered figure in Shi'ite Islam, and attracts millions of pilgrims each year from around the Muslim world.
Saudi Arabia has a sizeable Shi'ite Muslim minority, which has tense relations with the Sunni political and religious establishment, some members of which accuse them of being more loyal to Iran than to the kingdom.
The two countries are locked in a power struggle that has played out across the region and taken on a sectarian dimension. Each accuses the other of creating instability in the region.