Denver Democrats Michael Johnston and Pat Steadman were sworn in Friday.
Johnston of Mapleton Expeditionary School of the Arts in Thornton replaces former Senate President Peter Groff, who took an education post in Obama's administration.
Steadman helped lead a lawsuit challenging Amendment 2, which banned laws protecting gay people from discrimination. The U.S. Supreme Court struck down the law as unconstitutional in 1996. Steadman replaces Jennifer Veiga, who moved to Australia.
Johnston and Steadman were selected by vacancy committees this month.