Two miles (3.2 km) of the route through downtown were constructed in a cut-and-cover tunnel that became Broad Street, and the only underground portion of the subway.

The line was operated on a contract basis by New York State Railways from 1927 to 1938, and by the Rochester Transit Corporation (RTC) from 1938 until its closure.

The plans were supported by a feasibility study from engineer George F. Swain and promoted by Mayor Hiram Edgerton and other civic leaders.

[4] The canal bed was widened and deepened 5 feet (1.5 m) in all directions, which required the removal of 1,000,000 cubic yards (760,000 m3) of earth and dolomite.

Starting on the first day of operations in 1927, the Rochester and Eastern Rapid Railway connected at Rowlands and terminated at City Hall station.

[3]: 89 In 1929, a special subway–surface operation began using a ramp at Emerson station to connect with the Dewey Avenue line to provide rush-hour service to Kodak Park, a major employer in the city.

[3]: 45 In the aftermath of the Great Depression, New York State Railways fell into bankruptcy along with other railroads that operated interurban lines in the area.

[2]: 15  While the company was in receivership, New York State Railways continued to operate the subway on a contract basis with the city of Rochester.

[3]: 48, 61 During World War II, wartime rationing made the subway popular once again, and annual ridership peaked at over 5 million in 1946 and 1947.

Proposals to extend the line were briefly considered in the press, but ridership began declining again in 1948, and the city council made plans to abandon the subway and use its route for a connecting highway to the New York State Thruway instead.

Limited freight service operated by connecting railroads lasted on the western portion of the subway route from Court Street to General Motors until 1976, when the city of Rochester elected to fill the cut to eliminate maintenance on the numerous bridges.

The trolley car was loaned to other organizations and returned to the Rochester & Genesee Valley Railroad Museum in 1998, prior to restoration.

[11] Locomotive L-2 was rescued from a Rochester scrap yard in the 1970s, and has been set aside for a potential future restoration by the New York Museum of Transportation.

[13] Other proposals were made for repurposing the tunnel instead of filling it in, such as removing Broad Street and restoring the old canal or constructing an underground retail center.

In 2018, a plan dubbed ROC the Riverway was unveiled that proposes removing the Broad Street level of the aqueduct and partially re-flooding the former canal and subway bed on the aqueduct with water similar to what was done with the historic canals at Canalside in Buffalo, NY and incorporate walkways to connect the nearby Blue Cross Arena with the nearby convention center.