The TRC would now attempt to serve the subpoena directly on Botha at his Wilderness home, deputy TRC chairman Dr Alex Boraine said in a statement.
Botha's lawyer Ernst Penzhorn on Wednesday informed the TRC that he would be going to Zambia and that the subpoena could not be served through him.
Nor would it be acceptable for the subpoena to be delivered to his office.
This was in stark contrast to previous arrangements where Botha's initial subpoena had been served on Penzhorn's office, Boraine said.
"We can only assume that there has been a very considerable change of heart and that Mr Botha's lawyers and Mr Botha himself are not prepared to co-operate in any way with the commission."
However, the process of serving the subpoena would continue.
It would now be delivered to the commission's head office in Cape Town on Wednesday night, and a TRC investigator would travel to Botha's home in the Wilderness and attempt to serve it directly on Botha on Thursday, Boraine said.
"It is a matter of considerable regret that the process laid down in the Act, and which the TRC is bound to follow, has been made so difficult as a result of the lack of co-operation from Mr Botha and his lawyers."
Botha was subpoenaed to attend the TRC's first state security council hearing in Johannesburg earlier this month, along with fellow members of his former cabinet.
His testimony was postponed on the grounds of ill-health, and commission chairman Archbishop Desmond Tutu said then that the next round could take place at George, near Botha's home, to accommodate him.
However, after Botha indicated his unwillingness to attend, Tutu said the hearing would continue and if the former state president failed to appear he would be prosecuted in terms of the commission's founding legislation.
The Promotion of National Unity and Reconciliation Act allows for a fine or up to two years' jail for disobeying a subpoena.
TRC spokeswoman Christelle Terreblanche on Tuesday said the second subpoena had not been entirely necessary.
"But we understand he was unwilling, so we were underscoring the fact that he is still under subpoena."