Karzai stopped short of blaming the Pakistani government directly, but said the issue would be raised with neighbouring Pakistan.
On Thursday, a suicide bomber posing as a peace messenger wounded Afghanistan's intelligence chief, Asadullah Khalid, dealing a blow to a nascent reconciliation process.
"Of course we will be seeking clarification from Pakistan because we know this man who came in the name of a guest to meet Asadullah Khalid came from Pakistan. We know that for a fact," said Karzai.
"We will be firmly and clearly seeking clarification and asking for any information that they may have."
The Afghan Taliban claimed responsibility for the attack, although it often makes exaggerated claims about attacks on foreign troops or government targets.
Karzai said the militant group was not behind the attack in the heart of the capital, Kabul.
"Apparently the Taliban claimed responsibility like many other attacks but such a complicated attack and a bomb hidden inside his body, this is not Taliban work," Karzai said.
"It's a completely professional (job) ... Taliban cannot do that and there are bigger and professional hands involved in it."
Karzai said he would discuss the issue with Pakistani officials during a meeting in Turkey.
"This is a very important issue for us and we hope that the Pakistan government in this regard gives us accurate information and cooperates seriously, so the doubts we have end," he said.
Ties between Kabul and Islamabad have been strained by cross-border raids by militants groups and accusations that Pakistan's intelligence agency backs Afghan insurgent groups to advance its interests in the country.
Pakistan denies the accusations and says it is committed to helping bring peace to Afghanistan.
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