17β-hydroxysteroid dehydrogenase 3 (17β-HSD 3) deficiency is a rare cause of 46,XY disorders of sex development (DSD). At puberty, these patients experience a surge of androstenedione and also testosterone, leading to substantial virilization. The origin of testosterone synthesis in these patients remains elusive. We investigated the expression of the isoenzyme AKR1C3 (17β-HSD 5) in the testis and patient-derived genital skin fibroblasts (GSF) as well as the ability of GSF to synthesize testosterone. Supernatants of GSF cultures and serum samples of one patient before and after gonadectomy were analyzed by liquid and gas chromatography/mass spectrometry. The androgenic potential of GSF-derived supernatants was also assessed by androgen receptor-mediated transactivation of a reporter gene in transiently transfected Chinese hamster ovary cells. Although AKR1C3 is expressed both in the testes and in GSF, androstenedione is rapidly metabolized and is not synthesized to testosterone. The transactivation potential of GSF supernatants towards the androgen receptor is declining within 48 h. However, under testis-equivalent androstenedione concentration, testosterone can be synthesized in 17β-HSD 3-negative GSF. After gonadectomy, both androstenedione and testosterone decline rapidly in vivo. In 17β-HSD 3 deficiency, relevant amounts of testosterone are synthesized most probably through AKR1C3 in the testis and not peripherally in GSF.