To examine further the previously demonstrated synergism between prolactin and progesterone on preputial glands of hypophysectomized, ovariectomized, immature rats, their effects on squalene production were determined and compared with the ability of 17beta-hydroxy-5alpha-androstan-3-one (DHT) and prolactin to increase the amount of squalene in the preputial glands. Glands from progesterone-treated rats incubated in vitro with [14C]mevalonic acid incorporated radioactivity into squalene (identified by chromatographic mobility) more rapidly than glands from controls or prolactin-treated rats. Using the same in vitro system, glands from prolactin-treated rats incorporated more [14C]acetate into squalene than those from progesterone-treated animals. In addition, results showed that prolactin and DHT increased nonradioactive squalene (identified by mass spectral analysis) content in the gland while progesterone had no effect. It is proposed that prolactin increases preputial gland squalene content by enhancing synthesis of mevalonic acid, while progesterone increases incorporation of mevalonic acid into squalene.