The previous record of 249.4km per hour was set by American Andy Roddick in a Davis Cup semi-final in 2004.
The 2.08m Karlovic, making his return from achilles surgery, hit the monster serve in the fourth set of his doubles tie which he and teammate Ivan Dodig lost 6-3, 3-6, 5-7, 6-3, 6-4 to Germany's Christopher Kas and Philipp Petzschner.
Germany lead Croatia 2-1 going into Sunday's reverse singles rubbers.
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