Those wanting to continue south can do so only from the two lanes on the right.
A limited version of the split has been operating since early last week, when the agency made one of the lanes on the right available to southbound traffic for the first time.
That caused queues back to Esmonde Rd in Takapuna on the first day.
Traffic came to a halt as drivers dithered over which flyover lane to choose, even though southbound vehicles have still been able to use either lane.
Although the agency says it simplified its signs to make the choice more straightforward, some delays continued yesterday afternoon as traffic was affected by last-minute lane changing.
The agency hopes drivers will adjust to the new layout quickly next week, as it also intends opening a fifth southbound motorway lane and a bus lane in the same direction through St Marys Bay on Monday.
Even so, regional director Stephen Town says drivers should choose their correct lane before they cross the harbour bridge if possible."
"If they make the wrong choice, they will not be able to change lanes on the flyover because of the central barrier," he said.
The new configuration will leave only one mainstream motorway lane available for drivers going to the central city through the Fanshawe St exit, compared with two now.
One of the existing lanes will be dedicated to buses only from Monday.
But the Fanshawe exit will still branch out to four vehicle lanestowards its intersection with Beaumont St.
Despite the extra capacity, changes to Auckland's motorway network invariably cause initial delays, as was shown by the queues of traffic that stretched back beyond Otahuhu when two lanes of the Victoria Park motorway tunnel were opened in November.
The third and final lane will open in March, once the harbour bridge's movable lane barrier is extended to the northern end of the tunnel, to allow the Transport Agency to use "tidal flow" traffic management across the 11 motorway lanes in total through St Marys Bay.
That will mark the completion of the $406 million tunnel project, which has been built as the first of the Government's seven designated "roads of national significance".