In Act 5, Scene 1 of Macbeth, what is Lady Macbeth referring to when she says, "Out, damned spot! Out, I say!"
Lady Macbeth refers to the blood of King Duncan when she cries, "Out, damned spot! Out, I say!" She tries to wash away the guilt which now consumes her.
In Act V, Scene 1, it is a transformed Lady Macbeth that audiences witness. As this scene opens, a gentlewoman informs the physician that Lady Macbeth has been exhibiting some rather strange behavior: She rises from bed and sleepwalks to her chest; she unlocks it and takes out a paper upon which she writes. Then she reads it, seals it, and replaces it into the chest. After doing all this, she returns to bed, still fast asleep. When the doctor asks the gentlewoman what Lady Macbeth says, if anything, the woman refuses to repeat what she has heard because she has no witness to support her.
The doctor and gentlewoman both watch as Lady Macbeth washes her hands and then attempts to rub out a spot, ordering this spot out. Further, she seems to be talking to someone else as she asks why they should fear anything when no one can lay the guilt on them. Nevertheless, her overwhelming guilt causes her to add, "Yet who would have thought the old man to have had so much blood in him?" (5.1.28-29).
In this first scene of Act V, the imagery of blood reappears and reinforces the theme of the destruction wrought by unchecked ambition. This blood acts as a reminder of the heinous acts of Lord and Lady Macbeth, as well as their guilt. Ironically, earlier in the play after Macbeth has murdered King Duncan, Lady Macbeth has insisted that "a little water" could wash away the blood of the king. Now, she seems to realize that "all great Neptune's ocean [will not] wash this blood" (2.2.60) clean from their hands, and she is going mad as a result of her guilt.