Located in Deveselu in southern Romania, the missile interceptor station will help defend NATO members against the threat of short- and medium-range ballistic missiles, particularly from the Middle East, officials said.
"Today the United States and Romania make history in delivering this system to the NATO alliance," said US commander in Europe and Africa Mark Ferguson at an inauguration ceremony with NATO chief Jens Stoltenberg.
But Russia sees the missile system as a security threat right on its doorstep, despite the US and NATO insisting it is not aimed at undermining Moscow's defences.
Relations between NATO and Moscow have sharply deteriorated since Russia annexed Crimea from Ukraine in March 2014, sparking fears among other eastern European countries that they too could be the targets of Russian aggression.
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Stoltenberg said the missile installation "represents a significant increase in the capability to defend European allies against proliferation of ballistic missiles" as it becomes part of a broader NATO missile shield with an installation in Poland as well.
"The site in Romania as well as the one in Poland are not directed against Russia. The interceptors are too few and located too far south or too close to Russia to be able to intercept Russian intercontinental ballistic missiles," he said.
The Deveselu site will host a battery of SM-2 missile interceptors and will officially be integrated into the NATO missile shield at the bloc's summit in Warsaw in July.
Work on the site began in October 2013 and is thought to have cost USD 800 million (700 million euros).
The US ambassador to NATO, Douglas Lute, has described the activation of the missile system as a gesture of his country's commitment to Article Five by which all 28 NATO members pledge a one-for-all, all-for-one response to any military threat if a member invokes the treaty clause in the face of an attack.