Indonesian Siti Aisyah and Vietnamese Doan Thi Huong are accused of smearing Kim Jong Nam's face with the banned VX nerve agent at a crowded airport terminal in Kuala Lumpur on February 13. He died about 20 minutes later.
The women, who face a possible death penalty if convicted, say they were duped into thinking they were playing a harmless prank for a hidden-camera TV show.
Judge Azmi Ariffin estimated the trial will last for two months.
The women appeared in court wearing traditional Malay dresses, smiling at their lawyers and embassy officials. They were handcuffed as they were led to the dock.
The two women are the only suspects in custody in a killing that South Korea's spy agency said was part of a five-year plot by North Korean leader Kim Jong Un to kill a brother he reportedly never met. Malaysian police have said four North Korean suspects fled the country the same day Kim Jong Nam was killed.
North Korea has a history of ordering killings of people it views as threats to its regime. While Kim Jong Nam was not thought to be seeking influence, his status as eldest son in the current generation of North Korea's founding family could have made him appear to be a danger to his half brother's rule.
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