Benfica, who won the European Cup in 1961 and 1962 and have reached the final five times since then, face Dynamo Kyiv.
While Benfica coach Roger Schmidt worries whether Alex Grimaldo will recover from the dizzy spell that forced him off in Saturday's 1-0 league win at Casa Pia, the Ukrainians made clear they have weightier concerns.
The game will be in Lodz, where Dynamo are in exile following Russia's invasion of Ukraine.
When the squad returned to training in Poland after winning in Austria to eliminate Sturm Graz in the previous round, the club's website made clear that this is more than a football game.
The players, it said, "realise they keep fighting for the honour of the country by adding points to the UEFA rankings table."
"All Kyivans miss home and look forward to the return to Ukraine, but the fact that they give our nation and warriors positive emotions makes them leave emotions behind. They have a single goal to go through to the Champions League group stage."
The two clubs met in the group stage last season.
They drew 0-0 in Kyiv in the opening round, Dynamo's only point. The Portuguese side won 2-0 at home in the last round to edge Barcelona for second in the group.
Returning to the group stage this year offers a hefty reward.
Each club is guaranteed 15.64 million euros ($16.04 million) for taking part. They can also collect 2.8m euros for every group stage victory and 930,000 euros for a draw. Teams that miss out, drop to the Europa League group stage where they are guaranteed 3.63m euros.
PSV Eindhoven, who needed an extra-time goal to eliminate Monaco in the previous round, face Rangers.
Ruud van Nistelrooy, who replaced Schmidt as coach at the 1988 European champions, faces old international team-mate and club adversary Giovanni van Bronckhorst, in Glasgow on Tuesday.
- 'It's not easy'-
The two coaches were long-time Netherlands team-mates.
They played on opposite sides the last time the clubs met in the Champions League.
Rangers, with Van Bronckhorst in midfield, won home and away in the 1999 group stage. Van Nistelrooy scored PSV's goal at Ibrox as Rangers won 4-1.
"We are ready," declared van Bronckhorst after a 4-0 league win over St. Johnstone on Saturday.
"We have two days to prepare and make the players fresh again and make sure we have our game plan in mind and execute the game plan as best as possible."
"It is a big game for us."
The third former champions in action are 1991 winners Red Star Belgrade, who visit Macabbi Haifa on Wednesday.
The Israelis surged through the last round by scoring three times in the last 30 minutes for an eye-catching 4-0 second-leg victory at Olympiakos.
Red Star coach Dejan Stankovic, a Champions League winner playing for Inter Milan, told Serbian media he was impressed calling Macabbi "a quality team in every area. I couldn't fault with them."
"It won't be easy. It is not easy to enter the Champions League," he said.
Fixtures (1900 GMT unless stated)
Tuesday
Bodo/Glimt (NOR) v Dinamo Zagreb (CRO), FC Copenhagen (DEN) v Trabzonspor (TUR), Rangers (SCO) v PSV Eindhoven (NED)
Wednesday
Qarabag (AZE) v Viktoria Plzen (CZE) (1645), Dynamo Kyiv (UKR) v Benfica (POR), Maccabi Haifa (ISR) v Red Star Belgrade (SRB)