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Hypophysectomized adult male rats were treated twice daily for 3 days with 0·9% NaCl solution, 200 µg ovine prolactin, 5 µg ovine luteinizing hormone (LH), or 200 µg prolactin +5 µg LH, starting 18 days after the operation. Minced testes from these animals were incubated in Krebs—Ringer bicarbonate buffer containing [1-¹⁴C]acetate for 3 h at 37 °C. The conversion of acetate into testosterone was calculated. It was not changed by prolactin treatment, was increased by LH (P < 0·01), and further increased by prolactin + LH (P < 0·01) to approximately the level observed in intact adult controls. The amount of testosterone in some of the incubates was measured by gas liquid chromatography. It was not changed in prolactin-treated animals and was increased in LH-treated animals (P < 0·02 compared with hypophysectomized controls). Treatment with prolactin+LH resulted in an increase in testosterone above that after treatment with LH alone (P < 0·01) and to a value similar to that in intact adult males. The incorporation of acetate into esterified cholesterol was decreased (P < 0·05) in groups treated with LH or prolactin + LH in which testosterone synthesis was increased. The incorporation of acetate into free cholesterol was not changed by any of the treatments. These results demonstrate synergistic action of prolactin and LH on the synthesis of testosterone in vitro confirming our earlier suggestions.