Was it tested immediately before you took it apart, or was it X number of years ago before you took it apart it worked? If it had been in storage some time, I would suggest "exercising" the jumpers and headers in case they developed an oxidation film that's preventing correct operation.

Other than that, is the VGA card different? Especially on buses over 33Mhz, different VLB i/o cards and VGA cards interact/react in different ways to each other and may need extensive fiddling with wait states, slot position etc, for them to work together nicely.

Some BIOSes allow the disabling of COM and Parallel ports, check that hasn't happened.

Edit: Also while you're in CMOS setup, check if there's an ISA bus clock ratio that it hasn't been set to sysclock/2 which the ISA half of the VLB card where the serial is probably won't like, and possibly 1/3 may be a little much for some cards, but 1/4 should be safe.

Just throwing in an idea to rule out the obvious, check again the jumpers on the I/O card in case one fell while working on the PC the the port is now disabled or misconfigured.

Edit: Oh wait, does this

Tempus wrote on 2021-07-22, 15:38:

- Tried another I/O-card which is VLB
- Tried another I/O-card which is ISA

mean that you tried 2 other I/O cards on the PC and you still the mouse doesn't work? If so I'd suggest a good look at the BIOS, maybe even a reset settings to default

What sort of settings can make serial port not work in the BIOS? I have taken pictures of the BIOS. Maybe you can see what is wrong?

Cutemouse shows what is in picture 2 and the ISA VLB I/O card looks like picture 1

Tempus wrote on 2021-07-22, 15:38:

So here is what I've tried: […]
Show full quote

So here is what I've tried:

- Moved the current I/O-card to the other two available VLB-slots
- Tried another I/O-card which is VLB
- Tried another I/O-card which is ISA
- Tried another mouse which is serial
- Tried to remove everything but the video card and the I/O-controller
- Tried connecting just one hard drive and one floppy drive
- Made sure the mouse is working on a different computer

I don't know if I listed all the things I've tried, but these are those I remember at the moment.

I really hope someone can help me get to the bottom of this!!!

Just to check, the MS mouse and other serial mouse both worked ok in another computer? And don't work in this computer, even using different IO cards?

In the rebuild did you change the PSU? Along with the other suggestions, might be worth checking the -12V supply. Have you got access to a multimeter to measure if -12V is going to the ISA slots?

Problem seems to have been fixed. I swapped Gotek Floppy Emulator to be at the end of the FDD-cable and the floppy drive to be placed before the twist, and it seemed to have done something. The Gotek was acting weird mostly the whole time being the B-drive. I should have gone after that before. Not sure if it was the main cause, but at least it is working now.