Harrison was seeking to have his one-game suspension for misuse of his helmet in an Oct. 20 against Oakland overturned. It was his third "serious" rules violation in the past 16 games.
Instead, the Chargers (6-1) will be without their leader in the secondary for this week's game against the New York Jets (2-5).
Gene Washington, the NFL director of football operations, issued the disciplinary action, citing two previous infractions, the second of which came with a warning that future penalties of this type could result in suspension.
Harrison has been guilty of illegal hits twice this season, beginning with the spearing of Kansas City Chiefs running back Priest Holmes on Oct. 13. That came with a $12,500 fine.
The play in question from Harrison's latest infraction came on Oakland's first drive of the first quarter - a 3rd-and-4 from San Diego's 9. The play was ruled an incomplete pass and no foul was called.
"The play was not a foul, is not a foul and will never be a foul under the current NFL playing rules," Chargers Coach Marty Schottenheimer said. "Rodney Harrison loves the game and plays it the way it's supposed to be played."
In a letter to Harrison, he said, "You made no effort to tackle the player or break up the pass and instead engaged in what appears to be simply a gratuitous effort to punish your opponent after the pass to him has been deflected by your teammate."
Schottenheimer was confident the appeal would be successful.
Harrison also was fined last year for a forearm hit on wideout Eddie Kennison of the Denver Broncos. In October 2000, he was fined a full game check for a helmet-to-helmet hit against Raiders tight end Jeremy Brigham.
Washington notes that the NFL has placed a high priority on enforcing unnecessary roughness rules in reports to its teams and in its discussions with representatives of the NFL Players Association on playing rules.
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