Coenzyme F420 has been isolated from methane‐forming bacteria and determined to be an N‐(N‐L‐lactyl‐γ‐L‐glutamyl)‐L‐glutamic acid phosphodiester of 7,8‐didemethyl‐8‐hydroxy‐5‐deazariboflavin 5′‐phosphate. This and related polyglutamate forms are the first instances of natural deazaflavins that function as two‐electron carriers in hydrogenase systems.