The couple exchanged marriage vows in Malibu, Calif. in September 2003, and are the parents of 3-year-old twins fathered by an anonymous sperm donor.
"Melissa and Tammy Etheridge are saddened to announce that they are now separated," the singer's representative told UsMagazine.com.
"We ask for consideration and respect for our family as we go through this difficult period," the former couple added in a statement.
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