O'Brien tells Winston a couple of different things with regard to Room 101. The first time that Winston asks O'Brien about the room, O'Brien says that Winston (and everyone else) already knows what is in the room.
Later on, O'Brien clarifies his remark. He reminds Winston of the time that Winston asked before. But this time he explains further. He tells Winston that what is in Room 101 is the "worst thing in the world."
We are not surprised when we hear that because we have seen what the prisoners look like when they come back from Room 101 and we have seen what they act like when they are told they have to go there. It must be something pretty horrible.
At the end of Part 3, Chapter 2, Winston asks O'Brien what is in Room 101, to which he replies:
You know what is in Room 101, Winston. Everyone knows what is in Room 101.
This foreshadows Winston's time in Room 101 in Part Three, Chapter Five. Here, O'Brien elaborates by saying that the room contains the "worst thing in the world." He also acknowledges that for every individual, this "worst thing" can vary quite widely because our deepest fears are personal and subjective.
The fact that the party knows the deepest fear of every individual in Oceania is indicative of the extent of its control. It demonstrates the success of its surveillance methods, like the telescreen, and how this information is manipulated by the party for the purposes of total control and exploitation.
Thus, by using intelligence to create a complete picture of an individual, the party can turn Room 101 into that individual's idea of hell. This is what makes Room 101 so unique and also so successful.