Former Tory chancellor Lord Lamont says it is the “duty” of Remain supporters not to undermine the government over Brexit.
Lord Lamont urged peers not to attempt to add conditions to the government’s Brexit bill as it passes through the Lords.
But another ex-chancellor, Labour’s Lord Darling, said the government should not get a “blank cheque”.
Peers are expected to continue debating the draft legislation until midnight.
A record 190 members are scheduled to speak over the course of the two days.
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Urging peers to leave the bill unamended, Lord Lamont, who backed a Leave vote in June’s referendum, said the views of the Remain side should be taken into account – and added: “But equally I believe that those who voted to Remain have a duty not to undermine the government’s negotiating position.”
But Lord Darling told peers: “I do not accept this argument that from now on those of us on the Remain side should sit back and say nothing and simply give the government a blank cheque to proceed.”
He said this was impossible with so many “unanswered questions”.
Lib Dem Baroness Kramer said voters should have “the final word” on the Brexit deal in a referendum.
MPs have already backed the proposed law, authorising Mrs May to inform the EU of the UK’s intention to leave.
There is unlikely to be a formal vote at the end of Tuesday’s second reading debate.
The government does not have a majority in the Lords and opposition and crossbench peers are seeking guarantees about the rights of EU citizens in Britain and the role of parliament in scrutinising the process.
Mrs May has said she wants to invoke Article 50 of the 2009 Lisbon Treaty – the formal two-year mechanism by which a state must leave the EU – by the end of March, and the government has warned the House of Lords not to frustrate the process.
In an unusual move, the prime minister watched Monday’s opening proceedings in person.
Although amendments are not voted on at this stage, speeches are being closely watched for signs of the mood of peers on the two key ones of parliament having a “final meaningful vote” on the draft Brexit agreement – and guaranteeing the rights of EU citizens in the UK.
Detailed scrutiny of the bill at committee stage is due to take place on 27 February and 1 March. If the bill is not amended, then it could theoretically be approved by the Lords at Third Reading on 7 March, becoming law shortly afterwards.
If peers do make changes to the bill, it would put them on a collision course with MPs – who overwhelmingly passed the bill unaltered and would be expected to overturn any Lords amendments.
Although the Conservatives have the largest number of peers in the Lords, with 252 members, they are vulnerable to being outvoted if opposition peers – including 202 Labour peers and 102 Lib Dems – join forces. Much will hinge of the actions of the 178 crossbenchers – who are not aligned to any party.