The Vatican blamed Wednesday's short cancellation on a "minor" intestinal problem suffered by the 83-year-old pontiff.
There are now real fears over his health giving rise to speculation over his fitness to attend several major events next month - including the beatification of Mother Teresa of Calcutta.
"The Holy Father, following an intestinal indisposition which arose yesterday afternoon, will not be present at today's general audience on the advice of his personal doctor," Vatican spokesman Joaquin Navarro-Valls said in a brief statement.
The pope's top aide, Secretary of State Cardinal Angelo Sodano, presided at the audience and read the pope's prepared address to pilgrims who had come to see the pontiff.
Sodano asked the crowd to pray for the pope's "speedy recovery". He told them the pope was watching the audience on television and would later deliver the final blessing to them via an audio link with the residence where he is resting.
A Reuters' source said the pope still planned to move back to the Vatican from his summer residence on Friday afternoon and, health permitting, preside at a Vatican ceremony on Saturday.
Surgeons removed a section of the pope's intestine after a life-saving operation following an assassination attempt in 13 May 1981.
Pope John Paul suffers from Parkinson's disease and has to rely on a wheelchair or his now-famous 'popemobile' to get around.
October presents the ageing pontiff with one of his biggest challenges, staminawise, of the year when the 25th anniversary of his election is marked on 16 October.
Several days later he will beatify Mother Teresa of Calcutta, declaring the nun, who died in 1997 after a life serving the destitute and dying, one of the blessed.
The ceremony is a key step towards sainthood for Roman Catholics.