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"With all of the Fake News coming out of NBC and the Networks, at what point is it appropriate to challenge their License? Bad for country!" Trump said in a Wednesday tweet at 9:55 am ET.
Comcast trading volume jumped and the shares fell to their lows of the morning immediately following the tweet.
The stock closed 0.7 percent lower.
Trump's tweet could raise fears of higher government scrutiny on Comcast, although networks are not licensed by any organization. The Federal Communications Commission requires licenses for individual radio and television stations.
"The President is an incredible advocate of the First Amendment," White House Press Secretary Sarah Sanders said during an Oct. 5 briefing. "With the First Amendment, with those freedoms also come responsibilities. And you have a responsibility to tell the truth, to be accurate."
Comcast shares are up about 8.4 percent for the year. The stock fell sharply in early September after one of its executives said the company expects a large subscriber loss during the third quarter.
The White House declined further comment other than to refer CNBC to the tweet itself. NBC did not return a call for comment.
The tweet about network licenses followed Trump's 9:45 a.m., ET, tweet calling NBC News "fake" news for a story about U.S. nuclear arsenal. The early Wednesday NBC report, citing three officials, said Trump said this summer he would like a nearly "tenfold increase" in U.S. nuclear arsenal.
In response, a White House official speaking on the condition of anonymity told NBC that the nuclear arsenal was not a primary topic of the briefing this past summer. Pentagon spokesperson Dana White told NBC "the secretary of Defense has many closed sessions with the president and his Cabinet members. Those conversations are privileged."
As a real estate tycoon, Trump was host of the NBC reality TV show "The Apprentice."
Disclosure: Comcast is the owner of NBCUniversal, the parent company of CNBC and CNBC.com.
— CNBC's Peter Schacknow contributed to this report.