Ueno Zoological Gardens said female panda Shin Shin and male Ri Ri, both 11, mated after they were put in the same enclosure Monday morning. They normally live in separate pens.
Last week, the zoo removed the pair from public viewing after the female came into heat.
The breeding season for pandas is from February to May, and the window for conceiving is said to be just a few days.
In 2012, a cub was born to the pair but died six days later from pneumonia. In 2013, Shin Shin showed signs of being pregnant, but it was a false call.