He is best known for writing music based around only one pitch, altered in all manners through microtonal oscillations, harmonic allusions, and changes in timbre and dynamics, as paradigmatically exemplified in his revolutionary Quattro pezzi su una nota sola ("Four Pieces on a single note", 1959). This composition remains his most famous work and one of the few performed to significant recognition during his lifetime. His musical output, which encompassed all Western classical genres except scenic music, remained largely undiscovered even within contemporary musical circles during most of his life. Today, some of his music has gained popularity in certain postmodern composition circles, with pieces like his "Anahit" and his String Quartets rising to increased prominence.

Scelsi collaborated with American composers including John Cage, Morton Feldman and Earle Brown, as well as being a friend and a mentor to Alvin Curran. His work was a source of inspiration to Ennio Morricone's Gruppo di Improvvisazione di Nuova Consonanza, and his music influenced composers like Tristan Murail and Solange Ancona.

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