The neo-Classical (or perhaps more accurately, neo-Baroque) concerns of Hindemith's startling instrumental works of the 1920s converged with what Hindemith later described as a growing awareness of "the ethical imperatives of music and the moral obligations of the musician" in his opera Mathis der Maler. Hindemith was exploring the conflict faced by an artist in turbulent times: to honor one's obligation to the society in which he lives or to remain true only to the artistic ideals he espouses. In the story of German Renaissance painter Matthias Grünewald, Hindemith was able to comment on his own situation. His stance did not go unnoticed by the Nazis, and the symphony he extracted from the opera was premiered in Berlin in 1934 only upon the insistence of conductor Wilhelm Furtwängler. The opera itself was not heard until 1938, in Zurich, after Hindemith had left Germany.

Hindemith's inspiration for the opera came partly from Grünewald's masterpiece, the Isenheim Altarpiece, a paneled triptych whose structure is mirrored in the three-movement symphony. The first movement, "Angelic Concert," serves as the opera's overture and appears here intact. After serene, widely-spaced chords on strings, the horns introduce a theme based on the German folk song "Es sungen drei Engel" ("Three angels sang a sweet song"), which reaches a glowing climax, richly orchestrated. A faster theme introduced by solo flute and strings is given a lively contrapuntal treatment, with resourceful and highly colorful orchestration, a new neo-Romantic sound-world for Hindemith. The climax of the movement is in two parts: the folksong returns on horns, then fully fleshed out in brass; after a moment of calm, fugato strings gather the orchestra for the final brazen (and brazenly triadic!) chords, with strings chiming and bells and triangle shimmering.

The second movement is titled "Entombment," and in the opera is a brief intermezzo expressing Mathis' grief on the death of his daughter. In this spare elegy, harmonies based on fourths and fifths prevail, intervals that the composer often employed to express solemnity. Flute and oboe, over pizzicato strings, entwine in a tender lament; there is a brief outcry of grief, then a return to the quietude of the opening, with the flute offering a gesture of consolation.

The music for the lengthy finale is drawn from the opera's episode in which the temptation of St. Anthony (one of the Isenheim Altarpiece scenes) is likened to the temptations and trials of Mathis himself. A chromatic recitative for unison lower strings is the thematic foundation of the three episodes that follow, beginning with a fast section in galloping rhythm, suggesting relentless pursuit and hopeless flight. An unsettling, high trill in the violins introduces the next section, a sensuous melody for violas and cellos depicting the pleasures of the flesh offered (vainly) to St. Anthony. The turmoil of the first section returns and reaches a cadence, at which point strings begin the contrapuntally complex resolution based on the plainchant hymn "Lauda Sion salvatorem." Ringing "Alleluias" in the brass bring the symphony to a close.

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