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North Korea refused on Saturday to lift a ban on South Koreans accessing their companies in a joint industrial zone on the North side of the border.
Entry to the Seoul-funded Kaesong complex has been barred since Wednesday, as inter-Korean tensions have risen to their highest level for years.
A few trucks with supplies and raw materials had optimistically turned up early at the border crossing to Kaesong, but turned back after being told the North Korean side was still not allowing any people or vehicles to cross.
South Korean managers have warned that the blocking of raw materials and personnel movement could force them to shut their Kaesong operations in a matter of days.
Pyongyang has allowed those South Koreans still inside Kaesong to leave, and 100 were expected to cross back to the South on Saturday, leaving around 500 in the complex, the South's Unification Ministry said.
Korean Unification Minister Ryoo Kihl-Jae said Friday that Seoul would consider pulling its citizens out of Kaesong if their situation becomes untenable.
Pyongyang has already threatened to pull out its 53,000 workers hired by 123 South Korean companies there and shut the whole complex down.
Kaesong, which lies 10 kilometres (six miles) inside the North, was established in 2004 and is a crucial hard currency source for North Korea.