Mothwerwell, Hamilton Accies and Queen's Park will take part in the Reserve League when it returns for this coming season.
All three will take part in the 10-team Reserve League, and 12-club Reserve League Cup.
The league, which was paused in season 2019-20 due to the pandemic, is being reintroduced, following discussions with member clubs.
It will kick off on the Tuesday, September 6, where Hamilton host Livingston at New Douglas Park, while Motherwell visit Queen's Park at Lochinch Pavilion.
The first derby of the season takes place at East Kilbride FC's K-Park on Tuesday, October 4, when Motherwell host Hamilton.
Other teams in the Reserve League are Ayr United, Dundee, Dundee United, Hibs, Kilmarnock, Livingston and Queen of the South.
Matches take place on Tuesday afternoons, and clubs will play each other once across the season, which is scheduled to end on April 4.
SPFL chief executive Neil Doncaster said: "We are pleased to be able to offer our member clubs the opportunity to be able to play SPFL cinch Reserve League football again, after the League was originally paused due to the pandemic.
"We would also like to thank our partners at cinch for its support of the SPFL cinch Reserve League and its fantastic ongoing backing of the Scottish game.”
The draw and fixtures for the Reserve League Cup will be published on Tuesday, August 2.
Dunfermline Athletic and Partick Thistle will be added to the competition, but won't play in the Reserve League.
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