The MD-80s had already been grounded two weeks ago
American Airlines has cancelled more than 1,000 flights after grounding its MD-80 aircraft to conduct extra safety inspections on wiring.
The cancellations - almost half of the airline's schedule - came after about 450 flights were scrapped on Tuesday.
The Federal Aviation Administration (FAA) raised concerns about the wiring inspections two weeks ago.
The FAA has been clamping down on safety inspections and several airlines have been forced to ground planes.
American Airlines said there could be more cancellations after Wednesday as it continued to check its fleet of MD-80 aircraft.
"We continue to inspect every airplane to ensure we are in total agreement with the specifications of the directive," chief executive Gerard Arpey said in a statement.
"We will get back to a full schedule as quickly as possible."
By Wednesday morning, only 30 of its 300 affected planes were back in operation.
Southwest, Delta and United have also grounded some of their fleets to carry out inspections.
The FAA said that it had checked several American MD-80s and decided that the work carried out two weeks ago did not meet its standards.
The airline runs about 2,300 flights a day, more than a third of which use MD-80s, mainly on mid-range flights.
The FAA has been tightening up its inspections since admitting that it was too lax with Southwest Airlines last year.
The US House of Representatives' Transportation Committee discovered that fuselage cracks on Southwest planes had gone undetected due to missed inspections.
The Senate is to hold its latest set of hearings on airline maintenance and inspection on Thursday.