Playing again in front of the home crowd, the Colts faced a big test in their AFC South rivals, the Jacksonville Jaguars. Jacksonville scored first on a 4-yard touchdown run by QB Byron Leftwich. The Colts were not able to respond until early in the second quarter when WR/PR Terrence Wilkins returned a punt 82 yards for a touchdown. Indianapolis grabbed the lead for the first time in the game during the third quarter on QB Peyton Manning's 30-yard touchdown pass to TE Dallas Clark. In the fourth quarter, Manning increased Indianapolis' lead on an uncharacteristic two-yard touchdown run. That score put the Colts up 21-7. The Jaguars, though, would get to within a touchdown late in the game on Byron Leftwich's 7-yard TD pass to RB Maurice Jones-Drew. That score, however, proved to be the last of the game as Indianapolis hung on for the victory, and improved their record to 3-0. Quarterback Peyton Manning finished the game 14 of 31 with 219 passing yards for one touchdown pass, while Byron Leftwich finished 16 of 28 with 107 passing yards for one touchdown pass. Leftwich also threw two interceptions. RB Maurice Jones-Drew had 103 rushing yards for Jacksonville, and Marvin Harrison had 6 catches for 94 yards receiving. Jacksonville Kicker Josh Scobee missed both of his field goals, and they proved crucial in the game in the late stages.

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