Rice, in Bucharest on the second leg of a four-nation European tour, signed the deal on Tuesday with Foreign Minister Razvan Ungureanu at a public ceremony in the Romanian capital.
"This agreement brings Romania into the mainstream of global security," the country's President Traian Basescu said at a news conference alongside Rice.
According to Basescu, the deal will see the United States establish four permanent military bases, one of which, the Mihail Kogalniceanu air base in the southeast of the country, was used by US forces during the Iraq war.
The other three sites are a base at Babadag, also in the southeast, and in Smardan to the south and Cincu in the centre.
US State Department officials told reporters travelling with Rice that the bases would be "the first US military presence in a former country member of Warsaw Pact", referring to the Soviet-era military alliance linking Eastern European nations to the USSR.
"We do not plan a permanent presence of substantial combat forces," the official added.