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Testosterone synthesis was studied in Acomys cahirinus by incubation of its testis with radioactive substrates. In comparison with other rodents, the Acomys testis was found to have a lower 17β-hydroxysteroid oxidoreductase activity, whereas the activity of 20-hydroxysteroid oxidoreductase was high. The accumulation of 5-ene-3β-hydroxy metabolites suggested that biosynthesis proceeded preferentially by the 4-ene route. This was confirmed by the simultaneous incubation of [³H]5-ene-3β-hydroxysteroids and [¹⁴C]4-en-3-oxosteroids. The predominant route of biosynthesis would, therefore, appear to be: pregnenolone progesterone 17-hydroxyprogesterone androstenedione testosterone.