Problem with Mesa 5i25

A step enable output might work marginally/intermittently if the I/O pin was not setup properly
because it will have a weak pullup if not programmed to be an output.

If you are not using a 64bit OS or one of the newer Ethernet FPGA cards,
its not really necessary to update mesaflash

I tried to setup and use another channel on the db25 connector for the Y axis and it doesn't work.
I setup the file .hal to use the internal connector with an adaptateur like this one:
sgcdn.startech.com/005329/media/products...arge/PEX2PECP2.B.jpg
It works fine!
It seems that several I/O pins are out of service on the db25 connector of the 5i25 board.
My linear encoder is also out of service!

Are you sure the 5i25 firmware configuration matches the breakout board you are using?

You can damage 5I25 I/O pins (or parallel port pins for that matter) If you plug in or unplug the DB25 cable and a common ground does not already exist between your machine and the PC.

I used the firmware 5i25_dmmbob1x2.
You talk about static electricity? My lathe and the PC are connected to ground.

I also have many problem with my stepper motor:
 

Are you using a 5i25?

Could you describe some of your "many problems" ?