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A complementary DNA encoding the mouse homolog of the testicular relaxin-like factor was cloned from a subtractive complementary DNA library, comprising clones preferentially expressed in the testes of mutant w/wv mice. The predicted amino acid structure conforms with that for other members of the insulin, insulin-like growth factor, and relaxin hormone family, with A, B, and C (connecting) domains. Northern and in situ transcript hybridization showed that the gene is exclusively expressed at a high level in the Leydig cells of both mutant and wild type testes and is up-regulated at puberty. The level of the specific messenger RNA is such as to imply that this novel member of the insulin family represents a major secretory product of Leydig cells, with a potential to be a part of a novel systemic feedback loop to other organs, including the brain.