The top slot is the only one that will run at 16x. The 2nd(middle slot) will only run at 8x when populated and the 3rd(bottom slot(only runs at 4x). So technically the top slot will off the best performance, but(depending on what gpu your are using) there will be little to no difference between a 16x 3.0 slot and an 8x 3.0 slot.