Suspicions begin to arise shortly after the messy assassination of King Duncan. Immediately following the discovery of the king's demise, Malcolm and Donalbain meet privately and decide that they can no longer trust the circumstances of Macbeth's castle:
"To Ireland, I; our separated fortune
Shall keep us both the safer. Where we are(160)
There's daggers in men's smiles: the near in blood,
The nearer bloody" (II.iii. 159-162).
Donalbain's speech to his brother clearly reveals that he suspects someone in the castle, and while it is not yet clear to him that Macbeth is responsible for his father's death, Donalbain clearly demonstrates that he has lost his trust in Macbeth's ability to protect him and his brother from coming to harm within the castle. Both brothers wisely decide to flee.
By Act Four, scene, three, Malcolm confirms his distrust of Macbeth to MacDuff in England, naming Macbeth:
"This tyrant, whose sole name blisters our tongues,
Was once thought honest."
Macbeth's murderous deeds fully come to light in this scene, in which MacDuff learns of his family's death at the new king's hands. Act Four, scene three's dialogue between MacDuff and Malcolm truly reveals the depth of the two men's general distrust of their new king.