The software was allegedly installed in Jeep Grand Cherokees made between 2014 and 2016 and Dodge Ram 1500s.
The US government is suing car firm
Fiat Chrysler, alleging the firm sold vehicles with "defeat devices"
that helped them pass emissions tests.
The firm did not disclose the emissions control software to regulators, the Department of Justice (DoJ) alleged. The software enabled the vehicles to produce lower emissions during roadworthiness tests than they did when used under normal driving conditions.
Fiat said it would "defend itself vigorously" against the accusations.
In a statement Fiat Chrysler spokesman, Eric Mayne, said the firm would defend itself "particularly against any claims that the company engaged in any deliberate scheme to install defeat devices to cheat US emissions tests".
The DoJ filed the complaint on behalf of the US Environmental Protection Agency (EPA).
It alleges the software features were installed in about 104,000 Dodge Ram 1500 and Jeep Grand Cherokee vehicles, model years 2014, 2015 and 2016, and sold in the US.
Formal accusation
Fiat Chrysler last week announced updates to its emissions software programs. The firm said it would install the modified software into the older vehicles and new ones.The Italian American firm said it "believes this will address the agencies' concerns regarding the emissions software calibrations in those vehicles."
The dispute recalls the fight over devices installed in certain Volkswagen cars that were designed to bypass emissions tests.
The civil complaint, filed on Tuesday in Detroit, is the formal accusation that Fiat fitted defeat devises to some vehicles.
It follows the EPA's notification in January of its investigation into Fiat Chrysler's compliance with the Clean Air Act.
At the time, the agency said it was looking into whether the software devices would fall under "narrow exclusions" granted in some instances.
Since then, other countries, including the UK and France, have also said they were investigating the company.
Analysis: Theo Leggett, business correspondent
Just how serious is this for Fiat Chrysler?It isn't into Volkswagen territory yet, even though the Department of Justice has accused it of using defeat devices.
For a start, the number of vehicles is much smaller. But there's also the question of what is, and what isn't, a 'defeat device'.
Volkswagen admitted it couldn't make its cars clean enough to pass emissions tests AND enable them to perform well on the road. So it decided to cheat - and deliberately designed a software tool to do just that.
FCA has been accused of fitting software to its cars which wasn't disclosed to regulators, and which made emissions control systems perform in a different way on the road to how they worked in the laboratory.
BUT - carmakers do use a variety of software to regulate emissions control systems, to ensure that they only work at the correct temperature, for example. Otherwise parts of the system can be damaged.
Fiat Chrysler insists it didn't set out to cheat the testing process. It was not, it says, involved in any 'deliberate scheme'.
Nevertheless, it was informed of the Environmental Protection Agency's concerns earlier this year - and asked to provide an explanation.
It seems the authorities, so far at least, aren't satisfied with what they've heard.
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