The room portals take Isaac to is determined at the time they're created; if Isaac clears the room the portal would take him to, he will still be taken to that room if he uses it.
If every room has been explored, portals summoned by the familiar will do nothing and Isaac will jump up and down in them in a purely cosmetic effect. Same effect occurs if a portal is summoned in rooms that doesn't apper on map, as Mom's Heart room in Mausoleum/Gehenna II.
Portals can lead to Curse Rooms, forcing Isaac to take damage in order to leave.
Portals will persist after Isaac uses them or leaves the room, and will always lead to the same room.
Portals do not respect pathing rules, and can spawn on single-tile walkways and in front of doors, forcing Isaac to use them and preventing him from leaving the room through that door. This can cause a softlock depending on what door it blocks and where the portal leads.
Lil Portal bears a close resemblance to the Portal enemies.
If a portal is reencountered during the ascent, such as in a previously completed boss room, the game may crash if the destination the portal originally connected to no longer exists in the now altered floor layout.
If Lil Portal consumes a Black Heart in a Devil Room that is part of the room layout (not a reward or contents of a bag or a chest), the game crashes.
A portal can be created next to a door which can block off an entire section of the map and cause a soft lock.