A third child was injured by unknown means as the youths waited at the bus stop just west of downtown Louisville, authorities said.
A person in a car drove by and shot at the waiting students, some of whom were not injured, Louisville Metro Police Maj. Shannon Lauder said during a news conference. No suspects were in custody Wednesday afternoon.
Police put a photo of a dark gray Jeep SUV on social media and asked for the public's help in finding it. The vehicle had Illinois license tags, with the plate number BD91644.
Louisville Mayor Greg Fischer said the shooting violated a "sacred space."
"And a teen that should be at school today will not be there, and will never be there again," he said.
Fischer said the fatal shooting was the city’s 145th homicide this year.
Police were working with the FBI and other agencies in investigating the shooting, Chief Erika Shields said Wednesday morning. The chief suggested the shooting could be gang-related.
"The reality of it is, we are dealing with very difficult gang issue in this city," Shields said. "Many of our gang members go to these schools." She said she did not believe the victim was involved with gangs.
The bus stop near the city's downtown was for students of Eastern High School.
Jefferson County Schools Superintendent Marty Pollio called the incident "one of the most difficult mornings of my career." He said support is being provided to students and teachers at the school.
The bus arrived shortly after the shooting, which occurred around 6:30 a.m., school officials said.
Maj. Lauder said Louisville Police is "going to throw all of our resources at finding out who did this."