After having fixed the earlier glitches, the company was seeing some issues again and working to resolve them, spokeswoman Caroline Hubberstey said.
The problem was again mainly affecting deposits, although a few financial institutions also had problems sending funds.
Hubberstey said most people were able to send an Interac e-Transfer, but when the notification for deposit arrived at the receiver, the receiver was unable to deposit the funds.
Several people took to Twitter to express concerns that payments for such things as rent might not go through or that they might be hit with non-sufficient funds (NSF) charges.
Interac said it had received some concerns about fees but would be working with financial institutions to address them.
It's also working to confirm the root cause and working with its financial institution partners to ensure that clients do not incur any fees.
TD Bank said it wasn't aware of any issues with bill payments, and kept its customers informed on the Interac e-Transfer outage through Twitter and telephone banking.
The bank said its service had been fully restored.
A spokeswoman for Scotiabank said it had been told that Interac was working to resolve the issue and was keeping the bank informed.
"We know that convenience is of the utmost importance to our customers and apologize for the inconvenience," Scotiabank said.
The initial problem affected service across the country and was first noticed Tuesday morning.
Interac said its other services — Interac Debit, Interac Cash and Interac Online — were operating normally.
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