Aktuelles AVRStudio von Atmel laden, installieren.
Der Rest steht in der Hilfe unter STK500 Manual Firmware Upgrade:

STK500 Manual Firmware Upgrade
The current version of AVR Studio will automatically detect any old STK500 firmware version and request the user to upgrade the firmware. If for some reason the upgrade fails (the serial cable is detached during upgrade, the power goes out, ...) the next time the AVR Studio tries to connect to the STK500, the board will not be detected and the automatic upgrade procedure will not start.
Following is a procedure on how to manually upgrade a STK500 board. This procedure should work in all cases:

Power off the STK500
Push the 'PROGRAM' button (located near the AT45D021 dataflash) while powering up the STK500
Start AVR Studio and make sure there is a serial connection between the PC and the STK500 DSUB9 marked 'RS232 CTRL'
Start the program 'Avr Prog' located in the AVR Studio 'Tools' menu
Locate the firmware upgrade hex-file 'stk500.ebn' by pushing the 'Browse'-button in the 'Avr Prog Hex File'-window. The path for the 'stk500.ebn' for a normal AVR Studio installation is "C:\Program Files\Atmel\AVR Tools\STK500".
Push the 'Program'-button in the 'Avr Prog Flash'-window. A progress bar will now appear while showing additional information messages. Wait until the verify operation is finished.
Close the 'Avr Prog' program
Power off and on the STK500 PCB. The STK500 is now ready to be used with the new firmware.
Keine Ahnung, ob´s noch ne andere Möglichkeit gibt ohne AVRStudio.

Rainer