Workers at two Mercedes-Benz factories near Tuscaloosa, Ala., voted on Friday against joining the United Automobile Workers, a stunning blow to the union’s campaign to gain ground in the South, where it has traditionally been weak.

The defeat, based on an unofficial union tally, came after Kay Ivey, Alabama’s governor, and other Republican leaders argued that a pro-union vote would choke off the investment that has transformed the state into a major auto producer. Hyundai and Honda also have large factories in Alabama that the U.A.W. is trying to organize.

The vote took on national significance as a test of whether the U.A.W. could build on a string of recent victories and gain ground in a state whose elected officials have been hostile to organized labor. The union has said it wants to organize every automobile factory in the United States, expanding its membership to include the employees of companies like Toyota and Tesla.

But the loss at the Mercedes plants will almost surely slow down the union’s campaign and probably force it to do more spadework to secure the support of workers before seeking to hold elections at other auto plants. Union leaders will want to spend time figuring out how best to counter the messages and tactics of local lawmakers and company executives.

“It hurts to lose, no doubt,” Elizabeth Shuler, president of the A.F.L.-C.I.O., said on Friday. “But we see it not as a loss, but a temporary setback. Workers will persevere no matter what it takes.”

Auto executives and conservative lawmakers are also likely to closely study the vote at Mercedes to figure out the best approaches to fend off the U.A.W. and other unions in future contests and to deter union campaigns from the get-go.