In the desert Southwest of America during the waning days of the Old West, three gunmen two wearing long duster overcoats (Jack Elam, Woody Strode, Al Mulock) take over an isolated train depot and settle in to wait for the train. When the train finally comes, a nameless harmonica-playing stranger (Charles Bronson) gets off and asks for someone named Frank. They tell him Frank sent them in his place. In the ensuing showdown, all four men go down. Only the man with the harmonica gets up again.The soundtrack to the opening scene is a creative orchestration of ordinary sounds in the style of the John Cage. Composer Ennio Morricone uses dripping water, the clicking of a telegraph, a buzzing fly, and over all the persistent, annoying squeak of a windmill-powered pump to build tension, punctuate visual jokes, and emphasize the tedium of waiting for the train. The scant dialog allows the soundtrack to consume much more of our attention than a score usually does.

On a remote farm called Sweetwater, Brett McBain (Frank Wolff) and his family are preparing an outdoor wedding feast. McBain tells his son Patrick to drive into town to meet his new mother, who will be arriving by train from New Orleans. Suddenly shots ring out from the surrounding desert, and daughter Maureen, son Patrick, and McBain himself are slain. The youngest McBain, Timmy, runs out of the house to find that his entire family has been destroyed. He watches in terrified silence as a group of five gunmen in duster overcoats emerge from the scrub brush. When one of the men calls their leader Frank by name, asking what to do with the child, Frank (Henry Fonda) draws his pistol and slowly takes aim at the last remaining witness. With a self-satisfied grin, he pulls the trigger.

In the town of Flagstone, McBain's bride Jill (Claudia Cardinale) steps down from the train to find that no one is there to meet her. Giving up hope, she steps through the train station into the bustling new town still being built. She hires a carriage to drive her to Sweetwater. The farm's name draws laughter from the driver, Sam (Paolo Stoppa), who informs her that "Sweetwater" is a worthless piece of ground, and McBain is crazy for trying to farm it.

Along the way, Sam speeds through a group of railroad workers busily laying their "damn rails." Then he stops at a wayside inn/tavern/trading post, and Jill follows him inside. Her beauty draws the unwelcome attentions of the barman (Lionel Stander). After a noisy off-screen gun battle, the outlaw Cheyenne (Jason Robards) enters wearing shackles on his wrists. The sounds of a harmonica again reveal the presence of the nameless stranger, who has been watching from a dark corner of the tavern. Cheyenne dubs him "Harmonica," and he uses Harmonica's gun to force another patron to shoot apart the chain between his wrists. Cheyenne's men soon arrive, too late to help him escape the prison guards who now lie dead outside. Harmonica notes that the three men he killed. Wr