The accused Mexican kingpin Ismael "El Mayo" Zambada pleaded not guilty to drug trafficking charges in the same New York courthouse where fellow Sinaloa cartel co-founder Joaquin "El Chapo" Guzman was convicted five years earlier.
Zambada entered the plea to the 17 felony counts he faces, which also include money laundering and weapons charges, at a hearing before US Magistrate Judge James Cho in Brooklyn.
Judge Cho ordered that Zambada, 76, be jailed pending trial.
Prosecutor Francisco Navarro called Zambada "one of the most, if not the most, powerful narcotics kingpins in the world."
"A United States jail cell is the only thing that will prevent the defendant from committing further crimes and ensure his return to court," Mr Navarro told the hearing.
Defense lawyer Frank Perez did not object to prosecutors' request to jail Zambada.
Wearing a gray short-sleeve shirt, Zambada did not speak other than to answer "yes" or "no" to the judge's questions through an interpreter.
Zambada was taken into custody on 25 July at a New Mexico airfield along with one of Guzman's sons, Joaquin Guzman Lopez, in a major coup for US law enforcement.
He was then taken to El Paso, Texas, where he pleaded not guilty in federal court to separate drug trafficking charges.
US District Judge Kathleen Cardone last week had him transferred to Brooklyn after the US Department of Justice asked that he face trial there first.
Zambada was wheelchair bound for his first court appearance in El Paso, but walked on his own today.
"His health is very good," Mr Perez told reporters after the hearing.
The Brooklyn case began in 2009 and includes allegations related to the trafficking of fentanyl, a synthetic opioid fueling an epidemic in the United States.
Zambada is next due in court on 31 October.
"El Chapo" Guzman is serving a life sentence at a maximum security prison in Colorado.
His son has pleaded not guilty to drug trafficking charges in Chicago.
Shootouts this week in the western Mexican state of Sinaloa kindled fears that an intra-cartel war is about to break out following Zambada's arrest.
Fighting has killed 12 people since Monday, and yesterday state authorities canceled national day celebrations and shut schools in response to the escalating violence.
