I have a 2003 Ford E250 Super Cargo, When I am driving and the AC is on, there is a brief squeal every 5-8 seconds when driving over a certain speed, maybe 40 MPH. It does not do it while parked, which makes it difficult to locate. And it does not do it with AC off?

The serpentine belt may need replacing. I recommend replacing them every 80,000 miles. This would be the ideal time to also replace the tensioner and tensioner pulley.

Another possibility is the water pump. I replaced the water pump, on my 2000 Taurus, several months ago, because it developed an annoying whine. These noises can be especially difficult to isolate. When I tore the water pump apart, I could not even find anything wrong--it did solve the problem, however.

Worn serpentine belt or tensioner would prevent proper pulley contact, resulting in slip-stick noise each time the A/C clutch cycles.
The problem could also be with the A/C clutch contact surface. If it has become contaminated or corroded, each time the A/C cycles on you could hear noise.
If nothing else you've tried works, judicious application of electronics parts cleaner may resolve the issue.

The timing is so cosistant that it doesn't seem like belt or bearing.
From the drivers seat, it sounds like it is near brake master cylinder, This is not brake related.

I would recommend utilizing a set of Chassis Ears to pin-point the source of the sound.
Chassis Ear STE6600 - STE6600-AST
Available both wired and wireless.

Other potential sources could include vacuum actuators/mode doors.
Best of luck.

Thanks from you both,
Captn', I had thought about mics to locate problems in the past and now, glad to see a solution, thanks.

How's your truck lately?
Got it fixed yet?

How's it going Captn', Problem is still there. It's a strange one. Doesn't seem to increase with speed? Would think compressor bearing, idler, belt or something would increase with speed, Thanks.

I wonder if you may have a mysterious speed related wind whistle...
(grasping at straws now... )
Try masking off body seams and then removing the tape one area at a time till the noise comes back..? Just some thoughts.

It almost sounds like a bearing squeal, It definitely not wind, thanks again.

Definitely outside the cabin though? So it's not a mode door or actuator...

Don't make me come down there.. hehe

Unless cycling happens every few seconds, I will adjust Temperature see the effect.
I also feel like going to some of these problems people have, as it is easier to troubleshoot, than go back and forth. Thanks again
If ever in the Keys, stop by. Got Rum. Take Care