Note: Sometimes they won't appear so run away and come back. Or use a Tunnel to travel away and then back.
Also, if they die by some other means of your fists, do the same thing as above. They respawn.
You have to beat up (use must use your fists) 5 thirsty Harlequins. You will know if it counted because it says "X of 5 Harlequins beaten up." They are near the water, hence the thirsty part. So look along the river. 4 are in Centro District and 1 is in Campagna District.
I. The First Harlequin is in Centro District North and a bit west beside the river. He is between the Highest Doctor symbol on the map and the second Highest Tunnel Entrance. He is on some stairs going into the water.
II. The Second Harlequin is in Centro District North West beside the river again. He is to the East of the Bridge that goes to Vanticao District. Starting from the beginning of the bridge, if you use your map and go along the river East you will see a shaded area with a lighter circle in the middle. Go North of that circle and you will see a set of double stairs. He is on those stairs.
III. The Third Harlequin is in Centro District West beside the river. Go to ROSA IN FIORE
and go directly west from there. He is on a set of stairs again.
IV. The Fourth Harlequin is in Centro District South East under a bridge beside the river. Go to Ezio's Hideout and go DIRECTLY south of it. On your map you should see a Bridge that has lines going across it. He is under that bridge on the left side.
V. The Fifth and Last Harlequin is in Campagna District beside the Borgia Tower (Or Assassin's Tower) that is surrounded by water and cliffs. If you use your map and go West slightly from the Borgia/Assassin's Tower and then go slightly North, you will see a small rectangle that represents a small building. Go there and the Harlequin is behind the Small building along the edge of the cliff.