Disable one item in chekcedlistbox

Not sure i understand you well, but if you want to change item checked state based on condition: Define.Text != "Option 3", you can simplify your code to:

bool x = !(Define.Text == "Option 3");
checkedListBox1.SetItemChecked(5, x);

See: CheckedListBox.SetItemChecked Method (Int32, Boolean) (System.Windows.Forms)[^]

Seems, you're very beginner, so i'd strongly recommend to read this: 8 Most common mistakes C# developers make[^]

Your question is not clear and this answer will assume that you asked "How do I prevent a user changing the checkstate of a CheckedListBox item?".

It can be achieved via the ItemCheck event which occurs before the checkstate has changed. The ItemCheckEventArgs gives the index of the item which is about to change, the current checkstate in CurrentValue and the proposed new checkstate in NewValue. NewValue may be reassigned to change the behaviour of the control.

Changes to item 5 could be prevented like this

private bool lockItem5;

private void CheckedListBox_ItemCheck(object sender, ItemCheckEventArgs e) {
  CheckedListBox clbx = (CheckedListBox)sender;
  // The ItemCheck event is raised before the check is changed
  Debug.Print(@"""{0}"" Index:{1} Current:{2} New:{3}", clbx.Items[e.Index], e.Index, e.CurrentValue, e.NewValue);

  if (e.Index == 5 && lockItem5) {
    // Suppress the change by setting NewValue to CurrentValue
    e.NewValue = e.CurrentValue;
    Debug.Print("[{0}] change suppressed", e.Index);
  }
}

To give some visual indication that the item has been locked the checkstate could be set to CheckState.Indeterminate just before lockItem5 is set to true. That way item 5 will look different to the others and a user might realise that clicking repeatedly is not going to make it change!

Alan.